Facilitating assessment of blood flow and tissue perfusion using fluorescence-mediated photoplethysmography

ABSTRACT

Methods and systems for facilitating assessment of blood flow in a tissue volume of a subject are disclosed. In some variations, the method may include: after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light, acquiring fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume, estimating a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, and generating an assessment of blood flow in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent. The estimated molar concentration may be based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume of the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/433,502, filed Feb. 15, 2017, now U.S. Pat. No. 10,646,128, which claims priority to U.S. Provisional Application No. 62/296,006, filed Feb. 16, 2016, the entire contents of each of which are incorporated herein by reference.

FIELD

The present disclosure relates generally to the field of optical assessment of blood flow and/or tissue perfusion in tissue using photoplethysmography (PPG), and in particular to the quantitative assessment of blood flow and/or tissue perfusion in tissue.

BACKGROUND

Blood flow is a generic term used to define movement of blood through blood vessels, which can be quantified in terms such as volumetric flow rate (i.e., volume/time). Tissue perfusion is distinguished from vascular blood flow in that tissue perfusion defines movement of blood through blood vessels within a tissue volume. Tissue blood perfusion may be quantified in terms of volume/time/tissue volume (which may also be amount of blood/time/tissue amount (examples of “amount” include volume, area or mass)), though on occasion tissue mass is used instead of tissue volume. More specifically, tissue perfusion relates to the microcirculatory flow of blood per unit tissue volume in which oxygen and nutrients are provided to, and waste is removed from, the capillary bed of the tissue being perfused. Perfusion is associated with nutritive blood vessels (i.e., micro-vessels known as capillaries) that comprise the vessels associated with exchange of metabolites between blood and tissue, rather than larger diameter non-nutritive vessels. However, compared to blood movement through the larger diameter blood vessels, blood movement through individual capillaries can be highly erratic, principally due to vasomotion, wherein spontaneous oscillation in blood vessel tone manifests as pulsation in erythrocyte movement. In certain cases, for example, vasomotion can result in a temporary arrest of blood flow within the capillary bed for periods of up to 20 seconds, in order to facilitate oxygen diffusion from the individual erythrocytes through the capillary vessel wall and into adjacent tissue being perfused. Consequently, spontaneous oscillations in capillary blood flow can be independent of heart beat, innervation, or respiration. Such flow cannot be defined simply in terms of volume/time; instead, it must be characterized on the basis of the aggregate amount of blood in all the blood vessel (i.e., capillary) segments within a given volume of tissue. This characterization is reflected in the fact that all the measurements of capillary blood movement include a tissue volume-related dimension.

There are many circumstances in which medical practitioners and other clinicians desire to correctly assess blood flow and/or tissue perfusion in tissue. For example, in treating patients with wounded tissue, clinicians must correctly assess blood flow and/or tissue perfusion in and around a wound site, since poor tissue perfusion will have an adverse effect on the healing process. An accurate assessment of blood flow and/or tissue perfusion increases the chances of successful healing of both acute (e.g., surgical) and chronic wounds. The assessment of perfusion dynamics is also important in other clinical applications, such as pre-surgical evaluation of patients undergoing plastic reconstruction procedures (e.g., skin flap transfers), or assessment of viability and function of cardiac tissue during cardiac surgery (e.g., coronary artery bypass graft surgery, a partial left ventriculectomy or a left ventricular reduction via the Batista surgical procedure, etc.).

Quantification of tissue perfusion is of interest to clinicians across many surgical and non-surgical specialties. Although simple binary assessment (flow versus no-flow) may be adequate for some clinical applications, quantification of perfusion in standard measures is desirable in many other clinical applications. To date, quantitative assessment of tissue perfusion has remained elusive.

Photoplethysmography (PPG) is an optical technique that can be used to estimate changes in microvascular blood volume, and PPG-based technology has been deployed in commercially available medical devices for assessing pulse rate, oxygen saturation, blood pressure, and cardiac output. A typical output of such devices is the PPG waveform that corresponds to the heartbeat of the subject. PPG has not been utilized to provide measurements in standardized units when assessing blood flow. A PPG technology with such capabilities would enable routine measurements of blood flow in tissue, including perfusion measurements, to be made in standardized units of volume/unit time/tissue area. This would be of significant value to clinicians, as such measurements would allow direct inter-site and inter-subject comparisons.

SUMMARY

Described herein are systems and methods for facilitating assessment of blood flow and/or tissue perfusion in a tissue volume of a subject. In accordance with one aspect of the disclosure, the system may include one or more processors and memory having instructions stored thereon, wherein the instructions when executed by the one or more processors cause the system to: receive fluorescence data based on fluorescent light emitted from an excited fluorescence agent in the tissue volume, wherein the fluorescence agent in the tissue volume is excited after a predetermined amount of the fluorescence agent has been administered to the subject; estimate a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generate an assessment of blood flow and/or tissue perfusion in the tissue volume based at least in part on the time series of fluorescence input data and the estimated molar concentration of the fluorescence agent.

In some variations, the assessment of blood flow and/or tissue perfusion in the tissue volume may be based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow and/or tissue perfusion. In some of these variations, in generating an assessment of blood flow and/or tissue perfusion in the tissue volume, the processor may estimate the thickness increase of the blood volume layer based at least in part on the intensity of the fluorescence data during a diastolic phase of blood flow and the intensity of the fluorescence data during a systolic phase of blood flow and/or tissue perfusion. In some of these variations, the processor may estimate the thickness increase of the blood volume layer based at least in part on the estimated molar concentration of the fluorescence agent.

In some variations, the processor may estimate the circulating blood volume based at least in part on sex, body height, body weight, and/or other parameters associated with the subject or a population of subjects, including for example, parameters in connection with a co-morbid condition (e.g., diabetes). In some variations, the system may facilitate assessment of blood perfusion in the tissue volume. In some variations, the tissue volume may be selected in the fluorescence data by a user. In some variations, the fluorescent agent may comprise indocyanine green (ICG).

In some variations, the system may comprise a light source that excites the fluorescence agent in the tissue such that the fluorescence agent emits fluorescent light. In some variations, the system may comprise a sensor that acquires fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume. In some variations, the system may be or may include a portable hand-held system.

In accordance with one aspect of the disclosure, the method may include, after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light; acquiring fluorescence data based on the fluorescent light emitted during blood flow and/or tissue perfusion through the tissue volume; estimating a molar concentration of the fluorescence agent in the blood flowing through and/or perfusing the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generating an assessment of blood flow and/or tissue perfusion in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent. In some variations, the method may exclude the step of administration of the fluorescence agent to the subject.

In some variations, the assessment of blood flow and/or tissue perfusion in the tissue may be based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse. In some of these variations, generating an assessment of blood flow and/or tissue perfusion in the tissue volume may comprise estimating the thickness increase of the blood volume layer based at least in part on the intensity of the fluorescence data. In some of these variations, the assessment of blood flow in the tissue volume may be based at least in part on the intensity of the fluorescence data during a diastolic phase of blood flow and/or tissue perfusion and the intensity of the fluorescence data during a systolic phase of blood flow and/or tissue perfusion.

In some variations, the method may comprise estimating the circulating blood volume in the subject. In some of these variations, estimating a circulating blood volume in the subject may comprise estimating a circulating blood volume based at least in part on sex, body height, and body weight.

In some variations, the method may facilitate assessment of blood perfusion in the tissue volume. In some variations, the tissue volume may be selected in the fluorescence data by a user. In some variations, the fluorescence agent may be or may comprise indocyanine green.

In some variations, the systems and methods for estimating the circulating blood volume may be used in combination with or as means of additional data for use with quantitative determination of blood flow and/or tissue perfusion as described in more detail below. In some variations, there is provided a method for measuring a time-varying change in an amount of blood in a tissue volume. The method includes exciting a fluorescence agent such as for example indocyanine green (ICG) in the blood, acquiring a time-varying light intensity signal during a pulsatile flow of the blood through the tissue volume, wherein the pulsatile flow has a diastolic and a systolic phase resembling a conventional photoplethysmogram, and processing the acquired time-varying light intensity signal to obtain a measurement of the time-varying change in the amount of blood in the tissue volume.

In some variations, there is provided a system for measuring a time-varying change in an amount of blood in a tissue volume. The system includes a light source configured to excite a fluorescence agent such as for example ICG in the blood, a sensor configured to acquire a time-varying light intensity signal during a pulsatile flow of the blood through the tissue volume, wherein the pulsatile flow has a diastolic and a systolic phase resembling a conventional photoplethysmogram, and a processor configured to process the acquired time-varying light intensity signal to obtain a measurement of the time-varying change in the amount of blood in the tissue volume.

In some variations of the method and the system, a modified Beer Lambert law is applied at the diastolic and systolic phases of the pulsatile flow of blood through tissue volume such that ΔL=ln[(I_(e)Φ−I_(m)/I_(e)Φ−I_(p))](εC)⁻¹ where ΔL is a change in aggregate blood layer thickness within a given tissue volume, I_(e) is an intensity of an excitation light exciting the fluorescence agent in the blood, Φ is a quantum efficiency of the fluorescence agent, I_(m) is an intensity of the time-varying light intensity signal during the diastolic phase minimum of the pulsatile flow of the blood through the tissue volume, I_(p) is an intensity of the time-varying light intensity signal during the systolic phase maximum of the pulsatile flow of the blood through the tissue volume, c is a molar absorption coefficient for the fluorescence agent, and C is an estimated concentration of the fluorescence agent in the blood or an instantaneous molar concentration of the fluorescence agent in the blood.

In some variations of the method and the system, the instantaneous molar concentration of the fluorescence agent in the blood is determined by utilizing a concentration-mediated change in a fluorescence emission spectrum of the fluorescence agent. The concentration-mediated change in fluorescence emission spectrum of the fluorescence agent includes a monotonic spectral shift. In some variations, the concentration of the fluorescence agent in the blood may be estimated using parameters specific to the subject, population of subject or a combination thereof (e.g., body height, body weight, body blood volume, any other parameters relating to, for example, a systemic or local condition the subject or the population of subjects may have). The estimated concentration may be then used alone or in combination with the instantaneous molar concentration of the fluorescence agent in the Beer-Lambert law.

In various aspects of the method and the system, utilizing the concentration-mediated change in fluorescence emission spectrum of the fluorescence agent includes selecting first and second spectral bands of fluorescence emission spectrum of the fluorescence agent, acquiring first and second intensities of fluorescence emission integrated over wavelengths in the first and second spectral bands respectively, calculating a ratio of the first and second intensities, and deriving a value for C from the ratio. In various embodiments, the first spectral band includes wavelengths ranging from about 780 nm to about 835 nm, or a subset thereof, and the second spectral band includes wavelengths ranging from about 835 nm to about 1000 nm, or a subset thereof.

According to an embodiment, the first and second spectral bands are selected such that one of the first and second intensities varies monotonically with C, and one of the first and second intensities is unchanged with C. In another embodiment, the first and second spectral bands are selected such that the first and second intensities increase monotonically with C, but at different rates. In yet further embodiment, the first and second spectral bands are selected such that the first intensity increases monotonically with C, and the second intensity decreases monotonically with C. The instantaneous molar concentration of the fluorescence agent in blood ranges in various embodiments from about 2 μM to about 10 mM. In some variations, the value for C may be derived from the estimated concentration of the fluorescence agent based on the parameters of the subject or a population of subjects described above, and may be used alone or in combination with the experimentally derived value for C from the ratio.

In some embodiments, a system for facilitating assessment of blood flow in a tissue volume of a subject is provided, the system comprising: one or more processors; and memory having instructions stored thereon, wherein the instructions, when executed by the one or more processors, cause the system to: receive fluorescence data based on fluorescent light emitted from an excited fluorescence agent in the tissue volume, wherein the fluorescence agent in the tissue volume is excited after a predetermined amount of the fluorescence agent has been administered to the subject; estimate a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generate an assessment of blood flow in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent.

In some embodiments of the system, the assessment of blood flow in the tissue volume is based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.

In some embodiments of the system, generating an assessment of blood flow in the tissue volume comprises estimating the thickness increase of the blood volume layer based at least in part on an intensity of the fluorescence data during a diastolic phase of blood flow and the intensity of the fluorescence data during a systolic phase of blood flow.

In some embodiments of the system, the instructions cause the system to estimate the thickness increase of the blood volume layer based at least in part on the estimated molar concentration of the fluorescence agent.

In some embodiments of the system, wherein the instructions cause the system to estimate the circulating blood volume in the subject.

In some embodiments of the system, the instructions cause the system to estimate the circulating blood volume based at least in part on sex, body height, and body weight.

In some embodiments of the system, the system facilitates assessment of blood perfusion in the tissue volume.

In some embodiments of the system, the tissue volume is selected in the fluorescence data by a user.

In some embodiments of the system, the fluorescence agent comprises indocyanine green.

In some embodiments of the system, the system further comprises a light source that excites the fluorescence agent in the tissue volume such that the fluorescence agent emits the fluorescent light.

In some embodiments of the system, the system further comprises a sensor that acquires the fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume.

In some embodiments, a method for use in medical imaging for facilitating assessment of blood flow in a tissue volume of a subject is provided, the method comprising: after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light; acquiring fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume; estimating a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generating an assessment of blood flow in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent.

In some embodiments of the method, the assessment of blood flow in the tissue volume is based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.

In some embodiments of the method, generating an assessment of blood flow in the tissue volume comprises estimating the thickness increase of the blood volume layer based at least in part on an intensity of the fluorescence data.

In some embodiments of the method, the assessment of blood flow in the tissue volume is based at least in part on the intensity of the fluorescence data during a diastolic phase of blood flow and the intensity of the fluorescence data during a systolic phase of blood flow.

In some embodiments of the method, the estimated thickness increase of the blood volume layer is based at least in part on the estimated molar concentration of the fluorescence agent.

In some embodiments of the method, the method further comprises estimating the circulating blood volume in the subject.

In some embodiments of the method, estimating the circulating blood volume in the subject comprises estimating the circulating blood volume based at least in part on sex, body height, and body weight.

In some embodiments of the method, the method facilitates assessment of blood perfusion in the tissue volume.

In some embodiments of the method, the tissue volume is selected in the fluorescence data by a user.

In some embodiments of the method, the fluorescence agent comprises indocyanine green.

In some embodiments, a non-transitory computer-readable storage medium storing instructions for facilitating assessment of blood flow in a tissue volume of a subject is provided, wherein the instructions, when executed by one or more processors, cause a system to: receive fluorescence data based on fluorescent light emitted from an excited fluorescence agent in the tissue volume, wherein the fluorescence agent in the tissue volume is excited after a predetermined amount of the fluorescence agent has been administered to the subject; estimate a molar concentration of the fluorescence agent in the blood flowing through the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generate an assessment of blood flow in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent.

In some embodiments of the non-transitory computer-readable storage medium, the assessment of blood flow in the tissue volume is based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.

In some embodiments of the non-transitory computer-readable storage medium, generating an assessment of blood flow in the tissue volume comprises estimating the thickness increase of the blood volume layer based at least in part on an intensity of the fluorescence data during a diastolic phase of blood flow and the intensity of the fluorescence data during a systolic phase of blood flow.

In some embodiments of the non-transitory computer-readable storage medium, the instructions cause the system to estimate the thickness increase of the blood volume layer based at least in part on the estimated molar concentration of the fluorescence agent.

In some embodiments of the non-transitory computer-readable storage medium, the instructions cause the system to estimate the circulating blood volume in the subject.

In some embodiments of the non-transitory computer-readable storage medium, the instructions cause the system to estimate the circulating blood volume based at least in part on sex, body height, and body weight.

In some embodiments of the non-transitory computer-readable storage medium, the system facilitates assessment of blood perfusion in the tissue volume.

In some embodiments of the non-transitory computer-readable storage medium, the tissue volume is selected in the fluorescence data by a user.

In some embodiments of the non-transitory computer-readable storage medium, the fluorescence agent comprises indocyanine green.

In some embodiments, a kit is provided, the kit being for facilitating assessment of blood flow in a tissue volume of a subject and comprising any one of the systems described above in this section and a fluorescence imaging agent.

In some embodiments, a fluorescence imaging agent is provided, the agent being for use with any one of the systems described above in this section, any one of the methods described above in this section, any one of the nontransitory computer-readable storage mediums described above in this section, or the kit described above in this section for facilitating assessment of blood flow in a tissue volume of a subject.

It will be appreciated that any of the aspects of the disclosure can be combined. It will also be clear that all features and options mentioned can be combined.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B schematically illustrate the use of conventional photoplethysmography (PPG) in which a fingertip sensor is used to measure pulse rate, blood oxygen saturation or both.

FIG. 2 shows fluorescence emission spectra of indocyanine green (ICG) dye shifting to longer wavelengths with increasing molar concentration of the dye in blood according to an embodiment.

FIG. 3 illustrates an embodiment in which an instantaneous molar concentration of the fluorescence agent in the blood is determined by utilizing a spectral shift in the fluorescence emission spectrum of the fluorescence agent where first and second spectral bands are selected such that one of the first and second intensities varies monotonically with concentration, and one of the first and second intensities is unchanged with concentration.

FIG. 4 illustrates an embodiment in which an instantaneous molar concentration of the fluorescence agent in the blood is determined by utilizing a spectral shift in the fluorescence emission spectrum of the fluorescence agent where first and second spectral bands are selected such that the first and second intensities increase monotonically with concentration, but at different rates.

FIG. 5 illustrates an embodiment in which an instantaneous molar concentration of the fluorescence agent in the blood is determined by utilizing a spectral shift in the fluorescence emission spectrum of the fluorescence agent where first and second spectral bands are selected such that the first intensity increases monotonically with concentration, and the second intensity decreases monotonically with concentration.

FIG. 6 illustrates an example system for facilitating assessment of blood flow and/or perfusion in a tissue volume according to an embodiment.

FIG. 7 illustrates an example illumination module according to an embodiment.

FIG. 8 illustrates an example fluorescence emission acquisition module according to an embodiment.

FIG. 9 illustrates an example relationship between a ratio of ICG fluorescence intensities from a first spectral band ranging from about 820 to about 840 nm (where “SWL” denotes a short wavelength) and from the second spectral band ranging from about 840 nm to about 900 nm (where “LWL” denotes a long wavelength) and the instantaneous molar concentration of ICG.

FIG. 10 illustrates an alternative embodiment of the light source of the system in FIG. 6 .

FIGS. 11A, 11B, and 11C schematically illustrate the time-varying changes in an amount of blood in a tissue volume with phases of the cardiovascular pulse.

FIGS. 12A and 12B schematically represent the time-varying relationship between ICG concentration and fluorescence intensity during transit of ICG through a blood vessel.

FIGS. 13A and 13B show a fluorescence image of a tapered capillary tube containing a 0.025 mg/ml concentration ICG solution and a graph of the resulting linear relationship between capillary diameter and fluorescence intensity.

FIG. 14 is a plot of time-varying average fluorescence intensity emitted from an area of human forearm skin following injection of an ICG solution into the cubital vein.

FIG. 15 shows a train of PPG oscillations associated with a plot of fluorescence intensity emitted from a tapered capillary tube containing a 0.03 mg/ml concentration ICG solution as a function of capillary tube thickness.

FIG. 16 shows an example imaging system comprising an arrangement of optical components for implementing the FM-PPG methodology according to an embodiment.

FIG. 17 displays segments of time-varying average intensity graphs from certain working examples of the present disclosure contained herein.

FIG. 18 illustrates the effect of sample thickness on the example ratiometric calibration curves constructed for ICG in ethanol.

FIG. 19 illustrates the effect of sample thickness on the example ratiometric calibration curves constructed for ICG in human blood.

FIG. 20 illustrates a calibration curve interpolated from five data points;

FIG. 21 illustrates data for ICG in human blood.

FIG. 22 shows a comparison of the data generated using the FM-PPG example method and system from FIG. 14 with data for ICG in human blood.

FIG. 23 shows FM-PPG data from a Rhesus monkey eye, and in particular, for analysis of each sequence a time plot of image brightness (total intensity) vs. image number.

FIG. 24 shows FM-PPG data from a Rhesus monkey eye, and in particular, a plot generated for images 490-565, wherein the valleys between two consecutive blood flow pulses are selected (squares); the table indicates computed blood flow for each pulse as well as average flow.

FIG. 25 shows a second angiogram sequence in connection with FM-PPG data from a Rhesus monkey eye.

FIG. 26 shows a plot generated for images 300-381, wherein the valleys between five consecutive blood flow pulses are selected (squares); the table indicates computed blood flow for each pulse, as well as average flow.

FIG. 27 shows a third angiogram sequence in connection with FM-PPG data from a Rhesus monkey eye.

FIG. 28 shows a plot generated for images 260-290, wherein the valleys between two consecutive blood flow pulses are selected (squares); the table indicates computed blood flow for each pulse, as well as average flow.

FIGS. 29A and 29B show an image from the human eye angiogram (29A) and the retinal area represented as a box superimposed upon the autoradiograph of a flat-mounted choroid from the left eye of one of the monkeys (29B) used by Alm and Bill as described in the specification.

FIG. 30 is a schematic illustration of an exemplary imaging system for facilitating assessment of blood flow and/or perfusion in a tissue volume.

FIG. 31 is a schematic illustration of an illumination module in an exemplary imaging system for facilitating assessment of blood flow and/or perfusion in a tissue volume.

FIG. 32 is an illustrative timing diagram for visible and excitation illumination according to an exemplary imaging system for facilitating assessment of blood flow and/or perfusion in a tissue volume.

FIG. 33A illustrates a reference curve generated based on the absorptivity measurements of blood samples with known ICG concentrations. FIG. 33B illustrates control data, generated with use of the reference curve in FIG. 33A, for ICG concentration in the circulating blood volume over time following administration of a known amount of ICG to a subject.

FIG. 34A illustrates mean signal intensities for a short wavelength channel (SWL) and a long wavelength channel (LWL) over time following excitation of ICG administered to a subject.

FIG. 34B illustrates concentration of ICG over time as determined by a ratiometric method for determining fluorescence agent concentration.

FIG. 35 illustrates mean signal intensities for a short wavelength (SWL) channel and a long wavelength (LWL) channel over time following excitation of ICG administered to a subject, and concentration of ICG over time as determined by a ratiometric method for determining fluorescence agent concentration.

DETAILED DESCRIPTION

Reference will now be made in detail to implementations and various aspects and variations of the disclosure, examples of which are illustrated in the accompanying drawings. Various fluorescence imaging systems, methods, imaging agents, non-transitory computer-readable storage media, and kits are described herein. Although at least two variations of imaging systems, methods, imaging agents, non-transitory computer-readable storage media, and kits are described, other variations of fluorescence imaging systems, methods, imaging agents, non-transitory computer-readable storage media, and kits may include aspects of the systems, methods, imaging agents, non-transitory computer-readable storage media, and kits described herein combined in any suitable manner having combinations of all or some of the aspects described.

Conventional photoplethysmography (PPG) can estimate changes in tissue blood volume by detecting changes in the amount of red or near-infrared light transmitted through the tissue. As the blood volume within tissue expands and contracts during a cardiovascular pressure pulse corresponding to the heartbeat of the subject, the amount of light absorbed by the blood volume increases and decreases, respectively. As shown in FIGS. 1A and 1B, for example, the aggregate blood volume in the fingertip blood vessels is smallest during cardiovascular pressure pulse diastole and the volume is greatest during systole. Although it may be used for measuring pulse rate and blood oxygenation, this application of PPG technology is not configured to provide volumetric flow measurements in standardized units.

To be able to provide volumetric microvascular blood flow measurements in standardized units, the metrics of the PPG waveform must be related in a known and repeatable fashion to the blood volume changes in the tissue. It is possible to establish this type of deterministic relationship with the application of a modified Beer-Lambert law (also known as Beer's law, or the Beer-Lambert-Bouguer law). The Beer-Lambert law relates the attenuation of a light beam passing through a medium to the path length through the medium and its absorptivity and this relationship is utilized in conventional PPG. Conventional PPG is performed by passing a beam of near-IR wavelengths of light through tissue (e.g., a fingertip), but the need for trans-illumination of tissue significantly limits application of this method to the more general case of volumetric blood flow measurements in tissue. In some variations, the present disclosure utilizes a modified Beer-Lambert law to enable such blood flow measurements using fluorescent light wavelengths emitted by a fluorescence agent such as a fluorescence dye. Such a dye may, for example, be bound preferentially to blood plasma, thereby making it possible to aim both the light beam source and fluorescent light detector at the same aspect of a target tissue. The fluorescent light emitted from, for example, the dye-tagged plasma component of blood will conform to the modified Beer-Lambert law and, by solving the equation for the optical path length and quantifying the respective parameters, fluorescence-mediated PPG is capable of providing volumetric blood flow and/or tissue perfusion measurements without trans-illumination.

Thus, in contrast to the conventional PPG technology, the present disclosure provides fluorescence-mediated photoplethysmography (FM-PPG) for measuring time-varying changes in the amount of blood in a tissue volume, and presenting these changes as a blood flow and/or tissue perfusion (microvascular blood flow) in standardized units (e.g., volume/unit time). With FM-PPG, the detected fluorescence intensity is proportional to the instantaneous concentration of a fluorescence agent in the blood (e.g., a fluorescence agent in the blood plasma) to the estimated concentration of the fluorescence agent based on parameters specific to the subject or a population of subject as described in this specification, or a combination thereof, and can thus be utilized to determine blood flow in tissue, including microvascular blood flow or perfusion. Blood flow in tissue is generally understood as an increase in the total amount of blood flowing into an anatomic structure or region; blood flow encompasses tissue perfusion or microvascular blood flow, which is the amount of blood flowing through the capillaries of the vascular bed of the anatomic structure or region. In various embodiments, the methods and systems of the present disclosure are used for measuring blood flow in tissue, and more particularly, for measuring perfusion or microvascular blood flow in tissue. In some variations, the use of the method and system of the present disclosure includes the ability to discriminate between the blood flow and the microvascular blood flow.

FM-PPG may make routine quantification and other assessment of tissue blood perfusion clinically possible, using widely-accepted flow measurement dimensions (e.g., mL/sec*mm² for perfusion) that may be independent of the particular device from which they are obtained. Such quantification or other assessment of blood flow and/or tissue perfusion may be on an absolute scale and accordingly may provide widely-understood and recognized standard values suitable for routine clinical applications (e.g., intraoperative such as plastic surgery, reconstructive surgery, etc., or bedside use). Importantly, FM-PPG enables flow information at the tissue nutritive level in individual patients to be analyzed consistently and easily, similar to other known metrics like body temperature and blood pressure. For instance, FM-PPG may utilize perfusion for diagnostic purposes, to track patient health across different clinical assessment sessions, etc.

Methods for Facilitating Assessment of Blood Flow and/or Perfusion

In some variations, there are provided methods and systems for facilitating assessment of blood flow and/or tissue perfusion in a tissue volume of a subject. Described herein are multiple variations of methods for facilitating assessment of blood flow and/or perfusion, which may be used alone and/or in combination, in any suitable manner, for quantifying or otherwise assessing blood flow and/or perfusion in a tissue volume of a subject.

In some variations, a method for facilitating assessment of blood flow and/or tissue perfusion includes: after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light; acquiring fluorescence data based on the fluorescent light emitted during blood flow through the tissue volume; estimating a molar concentration of the fluorescence agent in the blood flowing through and/or perfusing the tissue volume, wherein the estimated molar concentration of the fluorescence agent is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject or a population of subjects; and/or generating an assessment of blood flow and/or tissue perfusion in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent.

In some variations, a suitable fluorescence agent is an agent which can circulate with the blood (e.g., an agent which can circulate with, for example, a component of the blood such as plasma in the blood) and which fluoresces when exposed to appropriate excitation light energy. Furthermore, in some variations, the fluorescence agent may exhibit a concentration-mediated change in its fluorescence emission spectrum. In various embodiments, the concentration-mediated change may include a monotonic spectral shift in the fluorescence emission spectrum of the fluorescence agent. An example of the fluorescence agent is a fluorescence dye, which includes any non-toxic fluorescence dye exhibiting a monotonic spectral shift with concentration. In certain variations, the fluorescence dye may include a dye that emits light in the near-infrared spectrum. In certain embodiments, the fluorescence dye may include a tricarbocyanine dye such as, for example, indocyanine green (ICG). In other variations the fluorescence dye may comprise fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, rose Bengal, trypan blue, fluoro-gold, green fluorescence protein, flavins (e.g., riboflavin, etc.), methylene blue, porphysomes, cyanine dyes (e.g., cathepsin-activated Cy5 combined with a targeting ligand, Cy5.5, etc.), IRDye800CW, CLR 1502 combined with a targeting ligand, OTL38 combined with a targeting ligand, or a combination thereof, which is excitable using excitation light wavelengths appropriate to each imaging agent. In some variations, an analogue or a derivative of the fluorescence imaging agent may be used. For example, a fluorescence dye analogue or a derivative may include a fluorescence dye that has been chemically modified, but still retains its ability to fluoresce when exposed to light energy of an appropriate wavelength. In variations in which some or all of the fluorescence is derived from autofluorescence, one or more of the fluorophores giving rise to the autofluorescence may be an endogenous tissue fluorophore (e.g., collagen, elastin, NADH, etc.), 5-aminolevulinic Acid (5-ΔLA), or a combination thereof.

In some variations, the method for facilitating assessment of blood flow and/or tissue perfusion comprises administering a fluorescence agent to the subject, such that the fluorescence agent circulates with the blood in the tissue volume as the blood flows through the tissue volume. In some variations, the fluorescence agent may be administered to the subject intravenously, e.g., as a bolus injection, in a suitable concentration for imaging. In some variations, the fluorescence agent may be injected into a vein, artery, microvasculature (e.g., a capillary bed) or a combination thereof of the subject such that it circulates in the microvasculature. In variations in which multiple fluorescence agents are used, such agents may be administered simultaneously, e.g. in a single bolus, or sequentially, e.g. in separate boluses. In some variations, the fluorescence agent may be administered by a catheter. In some variations, the fluorescence agent may be administered to the subject less than an hour in advance of performing the measurement according to the various embodiments. For example, the fluorescence agent may be administered to the subject less than 30 minutes in advance of the measurement. In yet other embodiments, the fluorescence agent may be administered at least 30 seconds in advance of performing the measurement. In still other variations, the fluorescence agent may be administered contemporaneously with performing the measurement as described in connection with the various embodiments. In some variations, the method may exclude the step of administration of the fluorescence agent to the subject.

The fluorescence agent may be provided as a lyophilized powder, solid, or liquid. In certain embodiments, the fluorescence agent may be provided in a vial (e.g., a sterile vial), which may permit reconstitution to a suitable concentration by administering a sterile fluid with a sterile syringe. Reconstitution may be performed using any appropriate carrier or diluent. For example, the fluorescence agent may be reconstituted with water immediately before administration. In various embodiments, any diluent or carrier which will maintain the fluorescence agent in solution may be used. As an example, in certain embodiments where the fluorescence agent is ICG, it may be reconstituted with water. In some embodiments, once the fluorescence agent is reconstituted, it may be mixed with additional diluents and carriers. In some variations, the fluorescence agent may be conjugated to another molecule, e.g., a protein, a peptide, an amino acid, a synthetic polymer, or a sugar e.g., to enhance solubility, stability, imaging properties or a combination thereof. Additional buffering agents may optionally be added including Tris, HCl, NaOH, phosphate buffer, HEPES.

In some variations, the fluorescence agent may be administered in various concentrations to achieve a desired circulating concentration in the blood. For example, when the fluorescence agent is ICG, it may be administered at a concentration of about 2.5 mg/mL to achieve a circulating concentration of about 5 μM to about 10 μM in blood. In various variations, the upper concentration limit for the administration of the fluorescence agent is the concentration at which the fluorescence agent becomes clinically toxic in circulating blood, and the lower concentration limit is the instrumental detection limit for detecting the fluorescence agent in circulating blood. In various other variations, the upper concentration limit for the administration of the fluorescence agent is the concentration at which the fluorescence agent becomes self-quenching. In some variations, a lower concentration limit for the administration of the fluorescence agent is the concentration at which the fluorescence agent becomes too difficult for conventional imaging technology to detect. For example, when the fluorescence agent is ICG, the circulating concentration of the fluorescence agent may range from 2 μM to about 10 mM.

As described herein in connection with the various embodiments, the fluorescence imaging agent may be used for blood flow imaging, tissue perfusion imaging, or a combination thereof, which may performed during an invasive surgical procedure, a minimally invasive surgical procedure, a non-invasive surgical procedure, or a combination thereof. Examples of invasive surgical procedures which may involve blood flow and tissue perfusion include a vascular procedure, a cardiac-related surgical procedure (e.g., CABG on pump or off pump) or a reconstructive surgical procedure. An example of a non-invasive or minimally invasive procedure includes wound (e.g., chronic wound such as for example pressure ulcers) treatment and/or management. In this regard, for example, a change in the wound over time, such as a change in wound dimensions (e.g., diameter, area), or a change in tissue perfusion in the wound and/or around the peri-wound, may be tracked over time with the application of the methods and systems. In some variations e.g, cardiac applications or other vascular applications, the imaging agent(s) (e.g., ICG alone or in combination with another imaging agent) may be injected intravenously through, for example, the central venous line, bypass pump and/or cardioplegia line to flow and/or perfuse the coronary vasculature, microvasculature and/or grafts. ICG may be administered as a dilute ICG/blood/saline solution down the grafted vessel such that, for example, the final concentration of ICG in the coronary artery or another vessel is approximately the same or lower as would result from injection of about 2.5 mg (i.e., 1 ml of 2.5 mg/ml) into the central line or the bypass pump. The ICG may be prepared by dissolving, for example, 25 mg of the solid in 10 ml sterile aqueous solvent, which may be provided with the ICG by the manufacturer. One milliliter of the ICG solution may be mixed with 500 ml of sterile saline (e.g., by injecting 1 ml of ICG into a 500 ml bag of saline). Thirty milliliters of the dilute ICG/saline solution may be added to 10 ml of the subject's blood, which may be obtained in an aseptic manner from the central arterial line or the bypass pump. ICG in blood binds to plasma proteins and facilitates preventing leakage out of the blood vessels. Mixing of ICG with blood may be performed using standard sterile techniques within the sterile surgical field. Ten milliliters of the ICG/saline/blood mixture may be administered for each graft or a vessel. Rather than administering ICG by injection through the wall of the graft or a vessel using a needle, ICG may be administered by means of a syringe attached to the (open) proximal end of the graft or a vessel. When the graft or a vessel is harvested surgeons routinely attach an adaptor to the proximal end of the graft or a vessel so that they can attach a saline filled syringe, seal off the distal end of the graft or a vessel and inject saline down the graft, pressurizing the graft or a vessel and thus assessing the integrity of the conduit (with respect to leaks, side branches etc.) prior to performing the first anastomosis.

Examples of suitable fluorescence agents administered are described in detail herein. However, the method may be used with any suitable kind of fluorescence agent administered to the subject in any suitable manner. The amount of fluorescence agent administered to the subject may be a predetermined and known amount (e.g., as measured in milligrams, etc.). Following administration of the fluorescence agent, the method may include exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light.

In some variations, the method may include acquiring fluorescence data based on the fluorescent light emitted from the fluorescence agent during blood flow and/or tissue perfusion through the tissue volume. For example, the method may include acquiring a time-varying fluorescent light intensity signal during the pulsatile flow of the blood through the tissue volume. The pulsatile flow may include a diastolic phase and a systolic phase. In some variations, the pulsatile flow arises from a cardiovascular pressure pulse, which may be generated by a heartbeat or simulated heartbeat (e.g., by using a blood pump). In some variations, acquiring fluorescence data may include operating a medical imaging system (e.g., similar to those described below) to excite the fluorescence agent and/or one or more other fluorophores and receive fluorescence light signals emitted from the excited fluorescent agent and/or one or more other fluorophores. In other variations, acquiring fluorescence data may include receiving fluorescence data (e.g., from a medical imaging device, from a data storage medium, etc.).

In some variations, the fluorescence data may include fluorescence intensity signal data representing the intensity of fluorescent light over time during blood flow and/or tissue perfusion through the tissue volume. In some variations, the fluorescence data may include a time series of fluorescence images including a plurality of individual image frames (e.g., fluorescence image frames), ordered consecutively by acquisition time. For example, acquiring fluorescence data may include acquiring a sequence of high-speed angiograms (e.g., at least approximately 20 frames per second) covering at least a portion of the transit of the fluorescence agent in the tissue volume. In some of these variations, the fluorescence images may be spatially registered (e.g., by aligning images based on ink dots applied to the skin of the subject prior to data acquisition). In some of these variations, the fluorescence images may be pre-processed for analysis (e.g., temporally cropping the fluorescence images to begin, for example, approximately 90 seconds after administration of the fluorescence agent).

In some variations, the method may include estimating a molar concentration of the fluorescence agent in the blood flowing through and/or perfusing the tissue volume, based on the predetermined amount of the fluorescence agent administered to the subject and an estimated circulating blood volume in the subject and/or a population of subjects. The molar concentration of the fluorescence agent in the blood flowing through and/or perfusing the tissue volume may be approximated by estimating the molar concentration of the fluorescence agent in the circulating blood volume of the subject and/or a population of subjects; for example, by dividing the predetermined amount of the administered fluorescence agent by the estimated circulating blood volume in the subject. After a certain amount of time has elapsed since administration of the fluorescence agent, the fluorescence agent has become thoroughly diluted in the subject's circulating blood volume. Assuming approximately uniform molar concentration of the fluorescence agent throughout the circulating blood volume of the subject (and within the blood flow and perfusion in the tissue volume), estimation of the molar concentration of the fluorescence agent in the circulating blood volume may also be an approximation of the molar concentration of the fluorescence agent in the blood flowing in and/or perfusing specifically the tissue volume (i.e., a steady state approximation).

The circulating blood volume in the subject may be estimated based on sex, body height, body weight, and/or any other suitable physical characteristics of the subject, and/or any suitable combinations thereof such as body mass index (BMI) alone or with other local or systemic parameters for the subject or a population of subjects (e.g., a subject having diabetes). In some instances, the circulating blood volume may be estimated applying an approximation formula or equation incorporating values for various one or more physical characteristics. In some instances, the circulating blood volume may be estimated by accessing a lookup table and/or data that provides estimated circulating blood volume based inputs of various one or more physical characteristics. However, the circulating blood volume may be estimated in any suitable manner.

In an exemplary embodiment, the circulating blood volume of the subject may be approximated by a method disclosed by Nadler et al. (Prediction of blood volume in normal human adults, Surgery 51:224-232 (1962): Man: BV=0.3669×h3+0.03219×w+0.6041 Woman: BV=0.3561×h3+0.03308×w+0.1833

-   -   and where:     -   h: Body height, in meters     -   w: Body weight, in kilograms     -   BV: Body Total Blood Volume, in liters

In some variations, the method may include generating an assessment of blood flow and/or perfusion in the tissue volume based at least in part on the fluorescence data and the estimated molar concentration of the fluorescence agent. The assessment of blood flow and/or perfusion in the tissue volume may be based on a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.

In some variations, the blood flow and/or perfusion may be assessed as F=(A) (ΔL) (P_(DC))/Δt, where A is the cross-sectional area of the tissue volume end, ΔL is the thickness increase of the blood volume layer, L, P_(DC) is pulse duty-cycle, and Δt=duration of a single pressure pulse. Derivation of this assessment is described as follows.

FIGS. 11A to 11C schematically illustrate the individual fluorescence agent-filled vessel segments within a rectangular volume of skin tissue, wherein the vessel segments are depicted during pressure pulse diastole (i.e., during a diastolic phase) and during the peak of systole (i.e., during a systolic phase). The arrows in these figures in connection with the tissue volumes (each having a cross-sectional area, A) indicate an increase in ICG fluorescence intensity that occurs when the diameters of the individual blood vessel segments increase as blood pressure rises from the diastolic to the systolic level during the diastolic and systolic phases of cardiovascular pulse, respectively. FIG. 11C schematically replicates the geometrical relationships depicted in FIG. 11B, except that the aggregate volumes of the individual blood vessel segments are represented by a single cubic volume. Additionally, the maximum amount of blood volume increase that occurs between the diastolic and systolic pressures is indicated as ΔV.

The total amount of blood flowing through the rectangular tissue volume during a single pressure pulse oscillation is proportional to the area beneath the pulse curve. If the pressure pulse were a square-wave, then the total volume flowing during a single pulse would be ΔV. However, the pressure pulse curve is not a square-wave, so the area under the actual pulse curve is a fraction of the square-wave area.

Therefore, the pulse duty-cycle (P_(DC)) may be defined as the fraction of the area under the square wave occupied by the area under the actual pulse curve. Thus, the actual blood flow or tissue perfusion through the tissue volume during one pressure pulse cycle, F, is represented by: F=(ΔV)(P _(DC))/Δt, where  (1) ΔV=(cross-sectional area of the tissue volume end, A)×(thickness increase, ΔL, of the blood volume layer, L), and Δt=duration of a single pressure pulse. Therefore, equation (1) may be restated as: F=(A)(ΔL)(P _(DC))/Δt  (2)

Absolute values can be determined for A, P_(DC), and Δt through, for instance, measurement of tissue volume and analysis of the signal and/or fluorescence data. Exemplary algorithm embodiments for determining, estimating, or otherwise assessing ΔL, and from which volumetric change in blood per unit time can be determined, are described below, including in the examples. Additional details and examples are described in U.S. patent application Ser. No. 14/305,950, filed Jun. 16, 2014 and titled “Quantification of Absolute Blood Flow in Tissue Using Fluorescence Mediated Photoplethysmography” and U.S. patent application Ser. No. 14/510,848, filed Oct. 9, 2014 and titled “Quantification of Absolute Blood Flow in Tissue Using Fluorescence Mediated Photoplethysmography,” both of which are incorporated in their entirety by this reference.

In some variations, generating an assessment of blood flow includes estimating the thickness increase of the blood volume layer ΔL based at least in part on the intensity of the fluorescence data. For instance, estimation of the thickness increase of the blood volume layer may involve application of a modified Beer-Lambert law for emitted fluorescence light. In particular, the modified Beer-Lambert law may be written as: ΔL=ln[(I _(e) Φ−I _(m))/(I _(e) Φ−I _(p))](εC)⁻¹  (3) wherein ΔL is a change in aggregate blood layer thickness within a given tissue volume, I_(e) is an intensity of an excitation light exciting the fluorescence agent, Φ is a quantum efficiency of the fluorescence agent, I_(m) is an intensity of the time-varying light intensity signal during the diastolic phase minimum of the pulsatile flow of the blood through the tissue volume, I_(p) is an intensity of the time-varying light intensity signal during the systolic phase maximum of the pulsatile flow of the blood through the tissue volume, ε is a molar absorption coefficient for the fluorescence agent, and C is the estimated molar concentration of the fluorescence agent in the blood. Further discussion of the modified Beer-Lambert Law, and exemplary applications thereof, are described in U.S. patent application Ser. Nos. 14/305,950 and 14/510,848 referenced above and incorporated in their entirety.

In some variations, the tissue volume may be defined in one of various manners. In one variation, the tissue volume may be defined as the entire field of view (FOV) that is encompassed by the fluorescence data (e.g., the FOV of the fluorescence images). In another variation, the tissue volume may be defined as a subset area of the FOV encompassed by the fluorescence data. For example, the tissue volume may be defined by a user based on selection on a user interface (e.g., clicking and dragging markers on a screen displaying the fluorescence data, etc.). As another example, the tissue volume may be defined based on the subset of pixels represented in the fluorescence data that exceed a predetermined signal intensity threshold (e.g., a baseline signal intensity corresponding to background or a tissue volume that is of less interest for analysis, etc.).

In some variations, there is provided a method for measuring a time-varying change in an amount of blood in a tissue volume. The method comprises exciting a fluorescence agent in the blood, acquiring a time-varying light intensity signal, which includes a time-varying fluorescence intensity signal, during a pulsatile flow of the blood through the tissue volume, the pulsatile flow having a diastolic phase and a systolic phase resembling a photoplethysmogram. The method further comprises processing the acquired time-varying light intensity signal to obtain a measurement of the time-varying change in the amount of blood in the tissue volume by applying a modified Beer-Lambert law at the diastolic and systolic phases.

In some variations, a method of measuring the time-varying change in the amount of blood in the tissue volume of the subject comprises administering the fluorescence agent to the subject such that the fluorescence agent circulates with the blood in the tissue volume as the blood flows through the tissue volume. Various examples of fluorescence agent administration, types and concentrations of fluorescence agents, are described in detail above and in the examples below. However, the method may be used with any suitable kind of fluorescence agent administered to the subject in any suitable manner.

The method for measuring the time-varying change in the amount of blood in the tissue volume further comprises acquiring the time-varying light intensity signal during the pulsatile flow of the blood through the tissue volume. Acquiring the time-varying light intensity signal may, in some variations, be similar to acquiring fluorescence data based on the fluorescent light emitted from the fluorescence agent during blood flow and/or tissue perfusion through the tissue volume, as described above. The method yet further comprises processing the acquired time-varying light intensity signal (e.g., a time-varying fluorescent light intensity signal) to provide a measurement of the time-varying change in the amount of blood in the tissue volume wherein a modified Beer-Lambert law is applied at the diastolic and systolic phases. The modified Beer-Lambert law for emitted fluorescence light may be written as: ΔL=ln[(I_(e)Φ−I_(m))/(I_(e)Φ−I_(p))](εC)⁻¹ wherein ΔL is a change in aggregate blood layer thickness within a given tissue volume, I_(e) is an intensity of an excitation light exciting the fluorescence agent, Φ is a quantum efficiency of the fluorescence agent, I_(m) is an intensity of the time-varying light intensity signal during the diastolic phase minimum of the pulsatile flow of the blood through the tissue volume, I_(p) is an intensity of the time-varying light intensity signal during the systolic phase maximum of the pulsatile flow of the blood through the tissue volume, ε is a molar absorption coefficient for the fluorescence agent, and C is an estimated concentration of the fluorescence agent as described in connection with various variations or an instantaneous molar concentration of the fluorescence agent in the blood, or a combination thereof.

As demonstrated in FIG. 2 , the emission spectrum for ICG dye in whole blood is different for each different molar concentration of the dye. In various embodiments, the instantaneous molar concentration of the fluorescence agent C is determined by utilizing a concentration-mediated change in a fluorescence emission spectrum of the fluorescence agent. The concentration-mediated change includes a monotonic spectral shift in the fluorescence emission spectrum of the fluorescence agent.

In some variations, utilizing the concentration-mediated change in the fluorescence emission spectrum of the fluorescence agent comprises selecting first and second spectral bands of a fluorescence emission spectrum of the fluorescence agent (e.g., as is shown in FIG. 3 ), acquiring first and second intensities of fluorescence emission integrated over wavelengths in the first and second spectral bands respectively, calculating a ratio of the first and second intensities, and deriving a value for C in the modified Beer-Lambert law from the calculated ratio.

In some variations, the first and second spectral bands may be selected in a number of ways. According to an embodiment, the first and second spectral bands are selected such that one of the first and second intensities varies (increases or decreases) monotonically with C, and one of the first and second intensities is unchanged with C. For example, as is illustrated in FIG. 3 , the intensity of fluorescence emission integrated over wavelengths for any bands selected in range B will remain nominally unchanged with increasing concentration of the fluorescence agent. Furthermore, the intensity of fluorescence emission integrated over wavelengths for any bands selected in range A will decrease with C. Consequently, the ratio of intensities of bands from A/B will decrease with C.

In some variations, the first and second spectral bands are selected such that the first and second intensities decrease monotonically with C, but at different rates. For example, as is illustrated in FIG. 4 , the intensity of fluorescence emission integrated over wavelengths for any bands selected in range B will decrease with C, but the intensity of fluorescence emission integrated over wavelengths for any bands selected in range A will decrease more slowly with C. Consequently, the ratio of intensities of bands from A/B will decrease with C.

In some variations, the first and second spectral bands are selected such that the first intensity increases monotonically with C, and the second intensity decreases monotonically with C. For example, as is illustrated in FIG. 5 , the intensity of fluorescence emission integrated over wavelengths for any bands selected in range B will increase with C, but the intensity of fluorescence emission integrated over wavelengths for any bands selected in range A will decrease with C. Consequently, the ratio of intensities of bands from A/B will decrease with C, but will do so at a greater rate than in the previous embodiments.

In some variations, the first spectral band comprises wavelengths ranging from about 780 nm to about 835 nm, or a subset thereof, and the second spectral band comprises wavelengths ranging from about 835 nm to about 1000 nm, or a subset thereof when, for example, the fluorescence agent is ICG.

By selecting the first and second spectral bands as described in connection with the various embodiments, a clinically discernible variation in the ratio is achieved over the range of clinically anticipated concentrations of the fluorescence agent in the circulating blood, and thus the instantaneous molar concentration, C, of the fluorescence agent can be determined.

In some variations, the method may further comprise correlating the measurement of the time-varying change in the amount of blood in the tissue volume to a biological parameter, a physiological parameter, a diagnostic parameter, a pathological parameter or a combination thereof. In an alternative embodiment, the method may comprise deriving a measurement of a change in a biological parameter, a physiological parameter, a diagnostic parameter, a pathological parameter or a combination thereof from the measurement of the time-varying change in the amount of blood in the tissue volume. In some variations, examples of the biological parameter, the physiological parameter, the diagnostic parameter, the pathological parameter or a combination thereof include those which are indicative or a certain condition of the tissue, a condition of the subject or a combination thereof (e.g., atherosclerosis, oxygenation, cardiac output).

The subject matter described herein facilitates differentiating between vessels (e.g., microvasculature and large vessels), and further facilitates the removal/suppression of noise and/or background. For example, in a post-acquisition image, contribution from a larger vessel may be suppressed in the image (e.g., contribution from a larger vessel passing through the field of view), which facilitates exclusion of non-nutritive blood vessels, and thus improves measurement and/or quantification of tissue perfusion arising from microvasculature.

Systems for Facilitating Assessment of Blood Flow and/or Perfusion

Described herein are multiple variations of systems for facilitating assessment of blood flow and/or tissue perfusion, which may be used alone and/or in combination, in any suitable manner, for quantifying or otherwise assessing blood flow and/or perfusion in a tissue volume of a subject. In some variations, there is provided a system for measuring the time-varying change in an amount of blood in the tissue volume and/or for generating and using the estimated concentration of the fluorescence agent. The system may comprise a light source configured to excite the fluorescence agent in the blood, a sensor configured to acquire the time-varying light intensity signal during the pulsatile flow of the blood through the tissue volume (where the pulsatile flow may be caused, for example, by a heartbeat or by means simulating the heartbeat such as, for example, a blood pump), the pulsatile flow having a diastolic and a systolic phase resembling a photoplethysmogram, and a processor configured to process the acquired time-varying light intensity signal to obtain a measurement of the time-varying change in the amount of blood in the tissue volume. A modified Beer-Lambert law is applied at the diastolic and systolic phases to obtain ΔL=ln[(I_(e) Φ−I_(m))/(I_(e) Φ−I_(p))](εC)⁻¹ as was described in connection with the method variations.

In some variations of the system, the concentration of the fluorescence agent, C, is determined based on a predetermined or known amount of the fluorescence agent administered to the subject and an estimated circulating blood volume in the subject.

In some variations of the system, the instantaneous molar concentration of the fluorescence agent, C, is determined by a utilization of a concentration-mediated change, including a monotonic spectral shift, in a fluorescence emission spectrum of the fluorescence agent, which may be used in combination with the estimated concentration of the fluorescence agent. In various embodiments, the utilization comprises a selection of first and second spectral bands of fluorescence emission spectrum of the fluorescence agent, an acquisition of first and second intensities of fluorescence emission integrated over wavelengths in the first and second spectral bands respectively, a calculation of a ratio of the first and second intensities, and a derivation of a value for C from the ratio.

In some variations, the selection of the first and second spectral bands is such that one of the first and second intensities varies monotonically with C, and one of the first and second intensities is unchanged with C. In some variations, the first and second intensities increase monotonically at different rates with C. In some variations, the first intensity increases monotonically with C, and the second intensity decreases monotonically with C. Examples relating to these embodiments are illustrated in FIGS. 3 to 5 . In various embodiments, the first spectral band comprises wavelengths ranging from about 780 nm to about 835 nm, or a subset thereof, and the second spectral band comprises wavelengths ranging from about 835 nm to about 1000 nm, or a subset thereof.

Referring to FIG. 6 , there is shown schematically an example system 610 for generation and use of the estimated concentration of the fluorescence agent in blood in the tissue volume, and/or measurement of the time-varying change in the amount of blood in the tissue volume. The system 10 comprises a light source 12 configured to excite the fluorescence agent 14 in the blood in the tissue volume, a sensor 16 configured to acquire the time-varying light intensity signal during the pulsatile flow of the blood through the tissue volume, and a processor assembly 18 configured to process the acquired time-varying light intensity signal to provide the measurement of the time-varying change in the amount of blood in the tissue volume, according to any of the variations of the methods described herein. The processor assembly 18 may include memory 68 with instructions thereon, a processor module 62 configured to execute the instructions on memory 68 to process the intensity signal as described in connection with the variations of the methods described herein, and a data storage module 64 to store the unprocessed and/or processed intensity signal. In some variations, the memory 68 and data storage module 64 may be embodied in the same storage medium, while in other variations the memory 68 and data storage module 64 may be embodied in different storage mediums. The system may further include a display 66 on which to display images and other data.

In some variations, the light source 12 comprises, for example, an illumination module 20 comprising a fluorescence excitation source configured to generate an excitation light having a suitable intensity and a suitable wavelength for exciting the fluorescence agent 14. FIG. 7 shows an example illumination module 20 according to an embodiment. The illumination module 20 comprises a laser diode 22 (e.g., which may comprise, for example, one or more fiber-coupled diode lasers) configured to provide an excitation light to excite the fluorescence agent 14 (not shown). Examples of other sources of the excitation light which may be used in various embodiments include one or more LEDs, arc lamps, or other illuminant technologies of sufficient intensity and appropriate wavelength to excite the fluorescence agent 14 in blood. For example, excitation of the fluorescence agent 14 in blood, wherein the fluorescence agent 14 is a fluorescence dye with near infra-red excitation and emission characteristics, may be performed using one or more 793 nm, conduction-cooled, single bar, fiber-coupled laser diode modules from DILAS Diode Laser Co, Germany.

In some variations, the light output from the light source 12 may be projected through one or more optical elements to shape and guide the output being used to illuminate the tissue area of interest. The shaping optics may consist of one or more lenses, light guides, and/or diffractive elements so as to ensure a flat field over substantially the entire field of view of the fluorescence emission acquisition module. The fluorescence excitation source may be selected to emit at a wavelength close to the absorption maximum of the fluorescence agent 14 (e.g., a fluorescence dye such as ICG, etc.). For example, as shown in FIG. 7 , the output 24 from the laser diode 22 may be passed through one or more focusing lenses 26, and then through a homogenizing light pipe 28 such as, for example, light pipes commonly available from Newport Corporation, USA. Finally, the light may be passed through an optical diffractive element 32 (e.g., one or more optical diffusers) such as, for example, ground glass diffractive elements also available from Newport Corporation, USA. Power to the laser diode 22 may be provided by, for example, a high-current laser driver such as those available from Lumina Power Inc. USA. The laser may be operated in a pulsed mode during the image acquisition process. In some variations, an optical sensor such as a solid state photodiode 30 may be incorporated into the illumination module 20 and may sample the illumination intensity produced by the illumination module 20 via scattered or defuse reflections from the various optical elements. In some variations, additional illumination sources may be used to provide guidance when aligning and positioning the module over the area of interest.

Referring back to FIG. 6 , in some variations, the sensor 16 comprises, for example, a fluorescence emission acquisition module configured to acquire a fluorescence signal (e.g., the time-varying light intensity signal) from the fluorescence agent 14, the fluorescence emission acquisition module comprising an image sensor. Referring to FIG. 8 , a fluorescence emission acquisition module 40 may be configured to acquire the fluorescence signal such as the time-varying light intensity signal from the fluorescence agent 14 (not shown). As is shown in FIG. 8 , the fluorescence emission 42 from the fluorescence agent 14 in blood (not shown) may be collected and focused onto a 2D solid state image sensor (e.g., an image sensor 44 and an image sensor 46) using a system of imaging optics 48 a, 48 b, and 48 c. The solid state image sensor may be a charge coupled device (CCD), a CMOS sensor, a CID or similar 2D sensor technology. An optical filter 50 (which may comprise a plurality of optical filters in various arrangements) is used to remove residual and reflected excitation light and to ensure that only the fluorescence emission is recorded at the image sensors 44 and 46. In some variations, a dichroic optical filter 52 is used to divide the fluorescence emission spectrum of the fluorescence agent 14 into two spectral channels (e.g., first and second spectral bands). In some variations, the dichroic optical filter 52 is designed such that the total fluorescence emission is divided generally equally between the two spectral channels, and such that the shorter wavelength channel collects light of wavelengths equal to or shorter than the fluorescence emission maximum, and the longer wavelength channel collects light equal to or longer than the fluorescence emission maximum. The charge that results from the optical signal transduced by the image sensors 44 and 46 is converted to an electrical video signal, which includes both digital and analog video signals, by the appropriate read-out and amplification electronics in the fluorescence emission acquisition module 40.

Although only two image sensors 44 and 46 are utilized in the embodiment shown in FIG. 8 , the preferred selection of the two spectral bands—where the fluorescence emission over wavelengths in one band monotonically increases with the fluorescence agent concentration and where the fluorescence emission integrated over wavelengths in another band monotonically decreases with the fluorescence agent concentration as shown in FIG. 5 —enables the possibility of utilizing the signals transduced by the two sensors in two beneficial ways. Firstly the signal from the two image sensors 44 and 46 may be combined to obtain the total fluorescence image signal intensity. This will enable the highest quality (lowest noise) fluorescent image to be generated. Secondly, the image signal from these two spectral bands can be ratioed on a pixel by pixel basis to determine the instantaneous molar concentration of fluorescence agent 14 in the blood. The molar concentration is an essential parameter in determining the time-varying change in the amount of blood in the tissue volume. The images from the two image sensors 44 and 46 show identical fields of view on a pixel by pixel basis. Furthermore, the range of variation of the ratio as shown in FIG. 9 , is increased and the determination of the instantaneous concentration of the fluorescence agent 14 is consequently more accurate by utilizing the selection of the spectral bands as is described in connection with the various embodiments.

Referring back to FIG. 6 , in some variations, the processor module 62 may be configured to analyze time-varying light intensity signals, perform calculations for the plethysmographic computation of the time-varying change in the amount of blood in the tissue volume, output the calculated information to an appropriate display and/or recording device, or a combination thereof. In various embodiments, the processor module 62 comprises any computer or computing means such as, for example, a tablet, laptop, desktop, networked computer, or dedicated standalone microprocessor. Inputs for the processor module 62 may be taken, for example, from the image sensors 44, 46 of the emission acquisition module 40 shown in FIG. 8 , from the solid state photodiode 30 in the illumination module 20 in FIG. 7 , and from any external control hardware such as a footswitch or remote-control. Output may be provided to the laser diode driver and optical alignment aids. In various embodiments, the processor module may have the capability to save image sequences to internal memory, such as a hard disk or flash memory, so as to enable post-processing of acquired data. In various embodiments, the processor module may have an internal clock to enable control of the various elements and ensure correct timing of illumination and sensor shutters. In various other embodiments, the processor module may also provide user input and graphical display of outputs.

In various other embodiments, the system 10 may additionally or alternatively comprise the light source 12A configured to excite the fluorescence agent 14 (not shown) as illustrated in FIG. 10 . The light source 12A comprises an illumination module comprising a first excitation source 90 and a second excitation source 92 configured to provide an excitation light to excite the fluorescence agent 14 (not shown). The output from each excitation source is passed through beam shaping and smoothing optics as described in connection with the previous embodiments. In some variations, the light source (not shown) comprises a fluorescence emission acquisition module consisting of fluorescence collecting and imaging optics similar to those described in connection with the previous embodiments, as well as an optical filter for rejection of residual and reflected excitation light (not shown). This system of optics may focus the collected fluorescence onto a single solid-state image sensor, which is read out by the processing module at each frame.

In operation, and with continuing reference to the embodiments in FIGS. 6 to 8 , the subject is positioned such that an area of interest is located beneath both the light source 12, for example comprising the illumination module 20, and the sensor 16, for example comprising the fluorescence emission acquisition module 40, of the system 10, and such that the illumination module 20 produces a substantially uniform field of illumination across substantially the entire area of interest. In some variations, prior to the administration of the fluorescence agent 14 to the subject, an image may be acquired of the area of interest for the purposes of background deduction. To acquire fluorescence images, the operator of the fluorescence imaging system 10 may initiate the image acquisition procedure by depressing a remote switch or foot-control, or via a keyboard (not shown) connected to the processor assembly 18 of the system 10 in FIG. 6 . As a result, the light source 12 (e.g., the laser diode 22 of the illumination module 20 in FIG. 7 ) is turned on and begins the shutter sequence for the image sensors (e.g., image sensors 44, 46 of the fluorescence emission acquisition module 40 in FIG. 8 ). When operating in the pulsed mode of the embodiment, each of the image sensors is read out simultaneously with the laser pulses. In this way, maximum fluorescence emission intensity is recorded, and signal-to-noise ratio may be optimized. In this embodiment, the fluorescence agent 14 is administered to the subject and delivered to the area of interest via arterial flow. Image acquisition is initiated, for example, shortly after administration of the fluorescence agent 14, and images of the fluorescence returned from substantially the entire area of interest are acquired throughout the ingress of the fluorescence agent 14. The fluorescence emission from the area of interest is collected by the front imaging optics of the fluorescence emission acquisition module 40. Residual and reflected excitation light is attenuated by the optical filters (e.g., optical filter 50 in FIG. 8 ).

In FIG. 8 , the dichroic optical filter 52 is used to divide the total fluorescence acquired into two selected spectral channels, as is described in connection with the various embodiments. In a single exposure, the images recorded by each sensor 44 and 46 are read out and sent to the processor module (not shown) of the processor 18 of the system 10 shown in FIG. 6 . In some variations, the processor module may perform averaging over adjacent pixels in each frame, as well as over multiple successive frames prior to performing any calculations of perfusion. The images recorded in each of the two spectral channels are compared, and the ratio of fluorescence intensity in each channel is calculated over a kernel of the field of view. The kernel may be a single pixel or an array of pixels in the field of view. Based on the calculated ratio, and on a previous calibration of the system, the concentration of ICG within the kernel is calculated. The combined signal from both image sensors 44 and 46 is then used, together with a measurement of the optical illumination intensity as measured by the sampling solid state photodiode 32 within the illumination module 20 in FIG. 7 to calculate the total fluorescence intensity, and determine the volume of blood in the kernel via an application of the modified Beer-Lambert law as is described. This processing is repeated over substantially the entire field of view, and the resulting measurement of perfusion and/or blood flow is displayed to the user on demand as, for example, a grayscale or false color image, or stored for later analysis.

In some variations, a system for facilitating assessment of blood flow and/or tissue perfusion in a tissue volume of a subject may be substantially similar to the system described above, except as described below. In some variations, a system for facilitating assessment of blood flow in a tissue volume of a subject includes one or more processors, and memory having instructions stored thereon. The instructions, when executed by the one or more processors, may cause the system to receive fluorescence data based on fluorescent light emitted from an excited fluorescence agent in the tissue volume, wherein the fluorescence agent in the tissue volume is excited after a predetermined amount of the fluorescence agent has been administered to the subject; estimate a molar concentration of the fluorescence agent in the blood flowing through and/or perfusing the tissue volume, wherein the estimated molar concentration is based on the predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject; and generate an assessment of blood flow in the tissue volume based at least in part on the time series of fluorescence input data and the estimated molar concentration of the fluorescence agent. In some variations, estimation of the molar concentration of the fluorescence agent and generation of the circulating blood volume in the subject are similar to the corresponding steps in the above-described method for facilitating assessment of blood flow and/or perfusion in a tissue volume of a subject.

In some variations, the system may be a hand-held imaging system. As shown in FIG. 30 , an exemplary hand-held system 3010 may include an illumination module 3011, an imaging module 3013, and a video processor/illuminator (VPI) box 3014. The VPI box 3014 may include an illumination source to provide illumination to the illumination module 3011 and a processor to receive data about light detected by the imaging module 3013 from a target 3012 illuminated by light output by the illumination module 3011. The illumination source may output light at different waveband regions (e.g., white (RGB) light, excitation light to induce fluorescence in the target 3012, etc.) depending on characteristics to be examined and the material of the target 3012. Light at different wavebands may be output by the illumination source simultaneously or sequentially. The VPI box 3014 may record, process, and/or display, etc., the resulting images and associated information.

In some variations, as shown in FIG. 31 , the illumination module 3011 may include at least two illumination ports directing illumination from an illumination source 3023, which may be included in the VPI box 3014, to a target (e.g., target field 3024). Each illumination port provides illumination over the target field, such that the light overlaps (e.g., substantially or completely) at the target material 3012 (shown in FIG. 30 ). The illumination distributions may be substantially similar and substantially overlap at the target 12 to provide substantially uniform illumination of the target. Each illumination port may include a lens module 3020, a connecting cable 3022 connected to the illumination light source 3023, and a light pipe 3021 adapting a high numerical aperture of the connecting cable 3022 to a lower numerical aperture of the lens module 3020. The lens module 3020 may be steerable (e.g., to simultaneously steer the first and second illumination ports through different fields of view).

In some variations, the VPI box 3014 may control illumination of a target and compensates for ambient light in a fluorescent signal generated by the target. For example, FIG. 32 illustrates a timing diagram for white light (RGB) and fluorescence excitation (Laser) illumination, and visible (VIS) and near-infrared fluorescence (FL) imaging sensor exposures configured to allow ambient room light subtraction from the fluorescence signal with a single sensor. Exposures of even (Exp 1) and odd (Exp 2) sensor pixel rows are shown interleaved with differing exposure times to facilitate isolation of an estimate of the ambient room light signal component. Pulsing the white light illumination at 80 Hz may bring the frequency of the flashing light above that which is perceptible by the human eye or which may trigger epileptic seizures. The visible light image exposure may be longer (e.g., twice) the RGB illumination to ensure overlap between the 60 Hz exposure frame rate and the 80 Hz RGB illumination pulse. Extra ambient light captured during the visible exposure may be ignored, due to the much greater intensity of the RGB illumination pulse and signal from the target.

By setting the NIR fluorescence image exposure times Exp 1 and Exp 2 to acquire for one half frame and one quarter frame periods, respectively, while running the excitation laser only in the last quarter of every third frame, the even rows (Exp 1) record one half frame of ambient room light in addition to one quarter frame of NIR fluorescence, while the odd rows (Exp 2) record one quarter frame of ambient room light plus one quarter frame of NIR fluorescence. Performing these fractional exposures within each visible or NIR fluorescence frame minimizes motion artifacts which would otherwise be caused by inserting additional exposure frames into the frame sequence for the purpose of ambient room light subtraction.

With such an acquisition design, an estimate of the ambient room light contribution to the image signals can be isolated by subtracting the Exp 2 sensor rows of the NIR fluorescence image from the Exp 1 sensor rows (interpolated to match Exp 2 pixel positions), yielding an estimate of one quarter frame of ambient room light signal. The estimate of one quarter frame of ambient room light signal can then be subtracted from the Exp 2 sensor rows of the NIR fluorescence image to yield an estimate of the NIR fluorescence signal with the one quarter frame of ambient room light removed. The control of the illumination and the exposure may be performed by the VPI box 3014.

In an exemplary embodiment of the ambient room light subtraction method, the signal output for Exp 1 sensor rows may be expressed as Exp 1=2A+F and the signal output for Exp 2 sensor rows may be expressed as Exp 2=A+F, where A=ambient light incident in one quarter frame period, and F=fluorescence incident in one quarter frame period. Solving for F yields F=2*Exp2−Exp1.

In some variations, the VPI box 3014 or other suitable processor in the hand-held system 3010 may further facilitate assessment of blood flow and/or perfusion based at least in part on a time series of fluorescence input data and the estimated molar concentration of the fluorescence agent, as further described above. In other variations, the VPI box 3014 or other suitable processor in the hand-held system 3010 may additionally or alternatively facilitate assessment of blood flow and/or perfusion based at least in part on of a concentration-mediated change in a fluorescence emission spectrum of the fluorescence agent, as further described above. In yet other variations, the VPI box 3014 or other suitable processor in the hand-held system 3010 may additionally or alternatively facilitate assessment of blood flow and/or perfusion based on any combination of the methods described herein, including in the example below.

In various embodiments, the system may be calibrated using known calibration methods and best available estimates for one or more parameters described herein in connection with the quantification. In various embodiments, the system may be calibrated from a theoretical value(s) using the best available numbers of the parameters described herein in connection with the quantification.

A kit may include any part of the systems described herein and a fluorescence agent such as, for example, a fluorescence dye such as ICG or any suitable fluorescence agent or a combination of fluorescence agents. In further aspects, a kit may include a tangible non-transitory computer readable medium having computer-executable (readable) program code embedded thereon that may provide instructions for causing one or more processors, when executing the instructions, to perform one or more of the methods for facilitating assessment of blood flow and/or perfusion in a tissue volume of a subject. The kit may include instructions for use of at least some of its components (e.g., for using the fluorescence agent, for installing the computer-executable (readable) program code with instructions embedded thereon, etc.). In yet further aspects, there is provided a fluorescence agent such as, for example, a fluorescence dye for use in the methods and systems described herein.

EXAMPLES Example #1: Determination of Thickness Increase of a Blood Volume Layer

In calculating blood flow as F=(A)(ΔL)(P_(DC))/Δt in Equation 1 above, one variation of calculating the blood volume layer thickness increase, or ΔL, may be based on concentration-dependent fluorescence quenching. Concentration-dependent fluorescence quenching displayed by a fluorescence dye (e.g. ICG) can be used to determine ΔL quantitatively in terms of length/time. Concentration-dependent fluorescence quenching is the phenomenon exhibited by certain dyes in solution, wherein the fluorescence intensity emitted by the solution increases along with dye concentration until a point is reached beyond which further concentration increase results in fluorescence diminution. For example, for ICG in blood, maximum fluorescence occurs at a concentration of 0.025 mg/ml (see Flower, R. W. and Hochheimer, B. F.: “Quantification of Indicator Dye Concentration in Ocular Blood Vessels”, Exp. Eye Res., Vol. 25: 103, August 1977); above or below this concentration, fluorescence diminution occurs fairly sharply, as shown in FIG. 12A, which schematically illustrates the relationship between ICG concentration and fluorescence intensity of ICG through a blood vessel. FIG. 12B schematically illustrates, for a fixed location within a blood vessel, the time-varying relationship between ICG concentration (solid curve) and fluorescence intensity (dashed curve) during transit of an ICG dye bolus through the vessel.

In particular, FIG. 12A shows the total fluorescence response as a function of increasing ICG concentration, and demonstrates that at high concentration, the agent is self-quenching. FIG. 12B demonstrates, in the solid line, the concentration during transit of a bolus of ICG. In the case of a large, concentrated bolus, the dotted line occurs, which shows the total fluorescence intensity can be quenched nearly to zero during the transit to give a double peaked fluorescence response as a function of time.

FIGS. 12A and 12B indicate the points A, C, and E at which dye concentration is 0.025 mg/ml, and the points B and D at which the concentration is at some significantly greater level at which fluorescence quenching takes place (e.g., in this example, about 10 times greater). FIG. 12B illustrates that as an ICG bolus transits the blood vessel and dye concentration increases (solid curve), ICG fluorescence also increases (dashed line) and reaches a maximum intensity when the dye concentration reaches 0.025 mg/ml (left-hand arrow C). As concentration continues to increase, fluorescence decreases due to concentration fluorescence quenching, reaching a minimum intensity (middle arrow D) when concentration reaches its maximum (about 0.250 in this example). Thereafter, concentration decreases, causing fluorescence intensity to increase, until it again reaches the maximum level of 0.025 mg/ml (right-hand arrow E); then as concentration continues to decrease, fluorescence also begins to decrease again.

The distinctive double peaks of equal maximum fluorescence intensity that occur during transit of an ICG bolus of sufficiently high concentration and volume integrity allow the determination of, in absolute terms, the increase in blood volume thickness, ΔL, depicted in FIG. 11C. Since the peaks occur at a concentration of 0.025 mg/ml, and since the fluorescence intensity emitted from a known area (A) at the precise time either peak occurs can be determined, the thickness of a layer of blood containing 0.025 mg/ml ICG that emits the same fluorescence intensity under identical conditions of illumination and magnification also can be empirically determined.

For example, using the same optical device and fluorescence excitation illumination used to acquire the high-speed angiographic images from which the intensity of the double-peaked fluorescence was obtained, a fluorescence image can be obtained of a finely tapered capillary tube filled with a 0.025 mg/ml ICG/blood or ICG/ethanol solution, as is illustrated, for example, in FIG. 13 . From this image (FIG. 13A), a graph of the linear relationship between capillary diameter, ΔL, and fluorescence intensity can be generated (FIG. 13B).

Absolute values are now known for all the terms in the equation F=(A×ΔL)/Δt, making it possible to solve for absolute blood flow through the volume of skin tissue lying beneath area A in terms of ml/sec. Therefore, this example illustrates absolute quantification of blood flow in a volume of tissue (e.g., skin blood flow) according to an embodiment.

Example #2: Determination of Thickness Increase of a Blood Volume Layer

In another example, following rapid cubital vein injection of 0.40 ml of 50 mg/ml aqueous ICG dye and an immediate, rapid 5.0-ml isotonic saline flush, high-spatial resolution angiographic images of a 250 mm² area of human medial contralateral forearm skin were obtained at the rate of 23/sec. The individual images in the angiogram sequence were re-registered to remove frame-to-frame arm movement, and from each of these images, average fluorescence intensity for the tissue area was calculated for each image. These data were then used to generate a plot of time-varying average fluorescence intensity, a portion of which (centered approximately about Image Number 100) is shown in FIG. 14 .

High-frequency PPG oscillations are clearly visible riding on the low-frequency component of the fluorescence intensity curve which is related to dye filling of the aggregate vascular volume contained within the volume of skin tissue, beneath the 250 mm² surface area. As is illustrated in FIG. 15 , three PPG oscillations (and a fraction of a fourth) centered about image number 100 have been juxtaposed beside a graph of the capillary diameter versus fluorescence data from FIG. 13B. Both graphs are on the same fluorescence intensity scale shown on the right of the figure.

An envelope defined by the minima (dashed line) and maxima (dashed line) of the PPG fluorescence-intensity oscillations is projected onto the fluorescence-intensity versus capillary-diameter graph and then onto the graph's abscissa. The width of the latter projection indicates the thickness of the blood-volume layer increase, ΔL=0.001 mm.

The average pulse duty-cycle (P_(DC)) for the three fluorescence-intensity oscillations in this example was determined to be 40%, and the average duration of one pulse was determined to be Δt=0.680 sec.

$\begin{matrix} {{{Since}F} = {(A)\left( {\Delta L} \right){\left( P_{DC} \right)/\Delta}t}} \\ {= {\left( {250{mm}^{2}} \right)\left( {0.001{mm}} \right){(0.4)/(0.68)}}} \\ {{= {0.147{mm}^{3}/\sec}},{{or}0.147{ml}/\sec}} \end{matrix}$ A very rough approximation can be made using the following average blood circulation and anatomical values:

${{{Total}{cardiac}{output}} = {{5L/\min} = {83.33{ml}/\sec}}}{{{Fraction}{of}{cardiac}{output}{to}{skin}} = {20\%{or}16.67{ml}/\sec}}\begin{matrix} {{{Skin}{area}\left( m^{2} \right)} = {\left\{ {\left\lbrack {{height}({in}) \times {weight}({lbs})} \right\rbrack/3131} \right\}{1/2}}} \\ {= {\left\{ {\left\lbrack {68 \times 154} \right\rbrack/3131} \right\}{1/2}}} \\ {= {1.83m^{2}}} \\ {= {1.83 \times 106{mm}^{2}}} \end{matrix}{{Therefore},{{{total}{blood}{flow}{to}1{mm}^{2}{of}{skin}} = {{\left( {16.67{ml}/\sec} \right)/\left( {1.83 \times 106} \right)} = {9.11 \times 10^{- 6}{ml}/\sec{Hence}}}},{{{blood}{flow}{to}250{mm}^{2}{of}{skin}} = {{250 \times 9.11 \times 10^{- 6}{ml}/\sec} = {0.0023{ml}/\sec}}}}$

This latter approximation of blood flow to 250 mm² of skin is about 60-times less than the 0.147 ml/sec derived using the fluorescence-mediated PPG method and system of the present disclosure as illustrated in the example above. However, such a discrepancy in results may be accounted for in view of the inherent assumption that blood flow is uniformly distributed throughout the entire body skin area in the calculation based on circulation/physiologic approximations, and that the capillary-diameter/fluorescence-intensity data in the FM-PPG example were compiled using ICG in ethanol rather than blood and by-eye capillary-diameter measurements were made with a superimposed scale rather than by digital means.

Therefore, this example illustrates that rapid venous injection of a small-volume, high-concentration dye bolus (e.g., ICG), followed by a saline flush to achieve a circulating peak dye concentration in excess of 38-μM, as required in the preceding calibration method, may be suitable for selected clinical implementations.

Example #3: Ratiometric Method for Fluorescence Agent Concentration Determination

An alternative calibration method was developed to accommodate the range of peak dye (e.g. ICG) concentrations encountered in routine clinical use. For example, typical ICG administration consists of injecting about 3 ml of 25 mg/ml aqueous solution, followed by a 5 ml saline flush. Injected dye bolus dilution from about 400 to 600 times occurs during intravascular transit to various sites of interest, resulting in a range of peak dye concentration of approximately 5.4- to 8-μM. To accommodate this variability of peak dye concentrations, additional fluorescence wavelength data is acquired simultaneously with the angiographic images; these data are used for dual-wavelength ratiometric analysis for determining intravascular dye concentration at the time each image is acquired.

Whereas the calibration method described earlier has the advantage of requiring no other data except an ICG fluorescence angiography sequence, the alternative calibration method has the advantage of being entirely transparent to the user, in that no deviation from the usual injection technique is required. However, the recording device's imaging optics is modified to permit continuous simultaneous measurement of two near IR wavelengths longer than those used for image formation.

As with the calibration method described earlier, in the alternative calibration method, note that the thickness increase ΔL, of the blood volume layer, L, depicted in FIG. 11C is embedded in the fluorescence generated by illumination of the aggregate ICG-dye tagged blood volume in the rectangular tissue volume during a single pressure pulse oscillation. When the surface area, A, of the rectangular tissue volume is illuminated with 805-nm wavelength laser light, the total fluorescence generated, F, is a function of excitation light intensity, i.e., the ICG molar absorption coefficient at 805-nm, £, the ICG molar concentration, C, the ICG quantum efficiency, Φ, and the aggregated ICG-tagged blood layer thickness, L. That is: F=f(I _(e) ,ε,C,Φ,L)  (4) However, taking into account that the excitation light is absorbed as it travels through ICG tagged blood volume, as described by the Beer-Lambert law of absorption, the intensity of emitted fluorescence light, If, is: If=I _(e)Φ(1−e ^(−εC L))  (5) Solving equation (3) for L: L=ln[I _(e)Φ/(I _(e) Φ−If)](εC)⁻¹  (6), wherein values for all the parameters are known, except for the instantaneous ICG molar concentration, C.

Dye (e.g. ICG) molar concentration can be determined by ratiometric analysis of, for example, two appropriate near-IR wavelengths above the band of wavelengths used to form the dye (e.g. ICG) fluorescence images.

Implementation of the ratiometric dye (e.g. ICG) molar concentration determination involves the addition of optical and electronic components into the fluorescence imaging pathway. An example of the insertion of these components, according to an embodiment, is shown schematically in FIG. 16 in the context of the SPY® imaging system by Novadaq Technologies Inc. of Mississauga, Canada. FIG. 16 shows schematically an imaging system 100 which comprises a CCD video camera 102 and an objective lens 104 for fluorescence imaging of fluorescence light from tissue 200. As is shown in the embodiment in FIG. 17 , the addition of optical and electronic components (e.g., component 106) into the fluorescence imaging pathway may comprise disposing such components between the SPY® objective lens 104 and the CCD video camera 102. Signal outputs from the two Si photo-detectors 108 and 110 shown in FIG. 16 are real-time analyzed, and the resultant molar concentration determined for each angiographic image will be embedded in its TIFF header and subsequently extracted as needed to determine the instantaneous dye molar concentration in the circulating blood. The embodiment in FIG. 16 is discussed in more detail below in connection with the example of hardware implementation.

The increase in thickness of the blood volume layer, ΔL, that occurs during a pressure pulse can be determined at any time during the angiographic sequence by using equation (4) to determine the layer thickness at the peak of a pressure pulse, L_(p), and at the pulse minimum, L_(m), and then calculating the difference between the two: ΔL=L _(p) −L _(m)=ln[(I _(e) Φ−I _(m))/(I _(e) Φ−I _(p))](εC)⁻¹  (7), where I_(p) and I_(m) are the respective fluorescence intensities measured at the pulse peak and minimum, and where L=ln[I _(e)Φ/(I _(e) Φ−I _(f))](εC)⁻¹, wherein,

-   -   L=aggregate thickness of blood layer in all vessels beneath area         A (cm)     -   I_(e)=excitation light intensity (W cm⁻²)     -   I_(f)=intensity of emitted fluorescence light (W cm⁻²)     -   ε=ICG molar absorption coefficient (M⁻¹ cm⁻¹)     -   Φ=ICG quantum efficiency (0.13)     -   A=area of interest (cm²)     -   C=ICG molar concentration (M)     -   L is determined at the systolic peak of the blood flow pulse         (L_(S)) and at its diastolic minimum (L_(D)), and then flow         through the tissue area of interest (A) is calculated as:         Flow=A(L _(S) −L _(D))/time,     -   and wherein, time is reckoned as the duration of the blood flow         pulse.

The fluorescence intensities I_(p) and I_(m) may be determined, for example, using the same data used in the example algorithm depicted in FIG. 15 , wherein the average intensity levels forming an envelope about the pressure pulses from the angiogram images in the shaded area in FIG. 14 are represented by the dashed horizontal lines. In the present example, however, those two horizontal are projected to the relative fluorescence intensity scale on the right-hand side of the graph; one line intercepts the ordinate at 1100, and the other line intercepts at 1035.

In order to convert these relative intensity levels for I_(p) and I_(m) to actual light intensity levels (μW/cm²), the CCD video camera was used as a light meter by calibrating the camera's gray scale output against controlled dye (e.g., ICG) fluorescence intensity input levels.

The calibration method was devised to take into account that the total fluorescence (I_(f), equation 3) is emitted spherically, and that only a fraction of it is detected by the camera, dependent upon the aperture diameter of the imaging system and the distance of the aperture from the emitting source. The camera's output was found to be linear from approximately 50- to 2500-μW, such that: I _(f)(μW)=(avg.intensity−100)/413.012  (8), therefore, I_(p)=2.905 μW, and I_(m)=2.749 μW. Noting that during acquisition of the angiogram data depicted in FIG. 11 , I_(e)=4.0 W, equation (5) becomes:

$\begin{matrix} {{\Delta L} = {{\ln\left\lbrack \frac{\begin{pmatrix} {\left( {4000 \times 10^{- 3} \times 0.13} \right) -} \\ \left( {{2.749 \times 10} - 3} \right) \end{pmatrix}}{\begin{pmatrix} {\left( {{4000 \times 10} - {3 \times 0.13}} \right) -} \\ \left( {2.905 \times 10^{- 3}} \right) \end{pmatrix}} \right\rbrack}\left( {\varepsilon C} \right)^{- 1}}} \\ {= {0.0005{mm}}} \end{matrix}$ Inserting this value for ΔL into equation (2):

$\begin{matrix} {F = {(A)\left( {\Delta L} \right){\left( P_{DC} \right)/\Delta}t}} \\ {= {\left( {250{mm}^{2}} \right)\left( {0.0005{mm}} \right){(0.4)/(0.68)}}} \\ {{= {0.0735{mm}^{3}/\sec}},{{or}0.0735{ml}/\sec}} \end{matrix}$

This blood flow (0.0735 ml/sec) is half that calculated by the previous method (0.147 ml/sec) used in the first example, making it closer to the approximation of 0.0023 ml/sec that was based on whole-body physiological parameters.

Variations in skin blood flow are induced by changes in a number of physiological parameters, as well as by changes in ambient temperature. To determine the magnitude of such variations, an experiment was performed in which two ICG angiograms of the same 250 mm² area of human forearm skin as in the example above were recorded within a period of 32 minutes, each following rapid injection of 0.33 ml of 25 mg/ml ICG and a 5 ml saline flush into the contralateral cubicle vein. The first angiogram was recorded at an ambient room temperature of about 70° F., and the second was recorded immediately after exposure for about 1 minute to radiation from a 24 W quartz halogen lamp at a distance of 6 inches; the temperature sensation was similar to that produced by rapid exhalation of breath through the mouth at a distance of several inches.

FIG. 17 shows segments of the time-varying average intensity graphs from the two experiments (top row) along with their respective first-derivative with respect to time graphs (bottom row). Since this experiment was performed prior to implementation of the calibration part of the FM-PPG algorithm, the calculated skin area blood flows at the bottom of the figure are only in relative terms, rather than the absolute terms of ml/sec. Nevertheless, the fact that blood flow was about 1.4 times greater at slightly elevated temperature, compared to ambient room temperature, is an indication of the range of variability in normal skin tissue that might be expected in FM-PPG blood flow measurements unless temperature and other physiological parameters at the time of data collection are not taken into account.

In this example, ICG was administered intravenously to a subject by injecting an ICG dye bolus of sufficiently high concentration that, during transit through the tissue site of interest, peak dye concentration would exceed 0.025 mg/ml (the maximum fluorescence intensity, and where concentration fluorescence quenching starts as described in more detail below). Under those conditions, the sub-sequence of angiogram images recording dye transit contained the information needed to determine ICG molar concentration for each image in the entire sequence as a function of image gray-scale intensity, assuming dye excitation level was constant throughout angiogram recording and that fluorescence-intensity vs. CCD camera gray-scale-output is known.

To counteract ICG dye fluorescence quenching, a real-time, two-wavelength ratiometric method for determination of ICG molar concentration in circulating blood was developed. It involves no special preparation, no deviation from a practitioner's usual regimen for dye injection, nor that the transit-phase of dye through the tissue of interest necessarily be recorded, but that fluorescence excitation intensity be maintained at a known constant level during image acquisition and that angiography not be performed when circulating blood is dye-saturated or that significant vascular staining has occurred. In various embodiments, implementation of the wavelength-ratiometric method involves additional optics and hardware and software for determining, simultaneously with each image acquired, the intensities of two bands of near-IR-wavelengths and for embedding these data in the corresponding image header. So far as angiogram acquisition is concerned, these additions and events are entirely transparent to the operator of any device incorporating them. In various embodiments, additional calibration steps are carried out on each device prior to its entering routine clinical use.

To evaluate the ratiometric method of ICG molar concentration, the ICG fluorescence imaging optical path of a FM-PPG system such as, for example, the imaging system 100 shown in FIG. 16 , was modified by insertion of a beam-splitter 106 between the objective lens 104 and CCD video camera 102 as is illustrated in FIG. 16 . In this example, the beam splitter 106 transmitted the band of ICG fluorescence wavelength(s) 112 used for image capture (approximately 815 to 845 nm wavelengths, which may be filtered by a bandpass filter 114) and diverted all higher wavelength(s) 116 (e.g., wavelengths >845 nm) to a parallel path (which may include a relay lens 118), along which the light was further divided by a second beam-splitter 120 into wavelengths less than approximately 875 nm (wavelength(s) 122) and wavelengths greater than approximately 875 nm (wavelength(s) 124). At the ends of these latter pathways were placed near-IR sensitive Si detectors (e.g., Si detectors 108 and 110) (Thorlabs, model PDA36A Si Switchable Gain Detector). Initially, narrow band-width filters centered about 850 (filter 126) and 900 nm (filter 128), respectively, were inserted in front of each of the detectors 108 and 110 to approximate the front-face-excitation fluorescence emission spectral data from whole blood samples containing 2-, 4-, 8-, 16-, and 32-μM ICG used to determine the two wavelengths on which the ratiometric method was based. A subsequent analysis indicated that in another variation, substantially the same ratiometric discrimination could be made using much wider bandwidths. Accordingly, in this embodiment, the narrow band-width filters were removed, thereby increasing the light intensity impinging on the Si detectors. It is noteworthy that the light impinging on each Si detector is reckoned to arise from the entire field of view recorded in each angiogram image. However, due to analysis equipment availability limitations, the post-secondary-beam-splitter light paths and focusing of light onto the Si detectors has not been rigorously verified.

In this example, each Si-detector amplifier output was connected to one of two input channels of a high-resolution digitizer (Advantech 10M, 12 bit, 4ch Simultaneous Analog Input Card, Model PCI-1714UL-BE), and the trigger output from the CCD video camera was connected to the digitizer's trigger input channel. Digitized outputs from each channel were inserted by the custom λ-link software into the header of each angiogram image recorded. The digitizer continuously acquired an aggregate of 500 k data samples per second, derived cyclically in equal portions from each of the three input channels. When custom λ-link software detected a rise above the 1.5-volt threshold in the camera trigger channel (signifying onset of a 5-msec pulse of excitation light from the 805-nm laser), data sample counting and recording from the other two channels started. The first 300 samples from the 850-nm channel were excluded (to avoid artifact associated with laser pulse rise time), the next 600 samples were recorded (empirically determined to be the optimal amount), and rest were excluded; the same acquisition algorithm was then applied to samples from the 900-nm channel. When the trigger channel voltage dropped below 1.5 volts at the end of the laser pulse, the program was primed to look for the next voltage rise above the threshold level.

The analysis part of the λ-link software was constructed that in the Phi-motion mode, intensity data from the 850- and 900-nm wavelength bands of light simultaneously embedded in each recorded angiogram image were extracted, producing two streams of digital voltage from the Si detector amplifiers. Each stream contained 600 data samples per excitation laser pulse (i.e., per image), so a total of 1,200 data samples were stored in each image header. The average level of the voltage samples from the 850-nm channel was divided by the average level of the samples from the 900-nm channel, producing a ratio that is transformed (via the calibration curve) into a μM-concentration of ICG (C) used to calculate absolute tissue perfusion.

The digitized outputs from the two channels, as well as the 2-wavelength ratio, were reported to 6 decimal places. It was empirically determined that the rolling average ratio from 40 consecutive images (the number recorded in approximately 1.7 seconds) was stable to 3 decimal places; therefore the last three are truncated for purposes of calculation and reporting.

The calibration curve associated with the example system is unique to the particular combination of parameters related to both its electrical and optical components (e.g., lens aperture, excitation laser power, Si-detector amplification, etc.), many of which have been optimized by empirical experimentation. Necessary characteristics of the curve are that it is monotonic, has a slope (positive or negative) sufficiently steep to permit adequate resolution for determining ICG concentration in blood, is reproducible for the given fixed set of device parameters, and is essentially independent of sample thickness.

Construction of the calibration curve for the system was based on samples of freshly acquired, anticoagulated whole human blood containing 2-, 4-, 8-, 12, and 16-μM concentrations of freshly reconstituted ICG dye. Three milliliters of each sample was placed in an open-top Petri dish, producing a 1.764-mm thick layer of blood with a large enough surface area to completely fill the device's field of view.

One by one, each of the five samples was positioned under the objective lens 104 of the imaging system 100, and a sequence of angiographic images approximately 5-sec long was recorded; this procedure was repeated twice more, as quickly as possible to avoid settling of the erythrocytes suspended in plasma. One-half milliliter of blood was removed from each sample, reducing the blood layer thickness by 0.294 mm, and again three sets of angiographic sequences of the five samples were recorded. Then an additional 0.5 ml of blood was removed from each sample, and the final three sets of angiographic sequences were recorded. Thus, nine sets of ratiometric data were acquired from each of the five ICG/blood samples, three sets each from three different sample thicknesses.

For purposes of comparison, identical sequences of ratiometric data were acquired from five samples of ICG in ethanol having a range of ICG concentrations identical to that of the ICG/blood samples. In this case, however, only one angiographic sequence instead of three was recorded from each of the five samples per sample layer thickness. This deviation from the ICG/blood protocol was necessitated by the fact that absorption of excitation light energy, especially with the higher ICG concentration samples, accelerated ethanol evaporation, thereby reducing sample layer thickness, as well as increasing sample concentration.

FIGS. 18 and 19 illustrate the effect of sample thickness on the 2-wavelength ratiometric calibration curves constructed for ICG in ethanol and in human blood, respectively. Although solutions of ICG in whole blood, serum, and ethanol exhibit similar behavior in terms of emitted fluorescence intensity as a function of ICG concentration, the curves in FIGS. 18 and 19 illustrate significantly different behavior between blood and ethanol solutions, especially at lower dye concentrations, in terms of the relationships between intensities of the bands of emitted wavelengths used to construct the 2-wavelength ratiometric calibration curves. A breakdown of the Beer-Lambert law related to differences in chemical interactions (e.g., dissociation and interaction) between ICG and the two solutes may account for this difference. The empirically derived calibration curve for ICG in blood meets the currently understood requisite characteristics as described above in this specification.

To use the calibration curve in conjunction with the software-embedded algorithm that calculates tissue perfusion, it has to be accessible in a digital format and appear to the software as a continuous function, even though, in this example, it is constructed from only five data points. This may be accomplished by finding an equation describing a smooth curve that passes through all five points. Unfortunately, no linear or second-order polynomial was found that fit satisfactorily, so a graph was manually constructed that does pass through all five points; and from it, twelve additional data points were derived, resulting in the curve in FIG. 20 . The software program interpolates between the total of 17 data points. The calibration curve is shown in FIG. 20 . As is shown in FIG. 21 , average variability of the ratio data along the curve averages +/−0.014.

Generation of the fluorescence data from human blood containing various concentrations of ICG used to determine the optimal wavelengths for construction of the ratiometric calibration curve was commissioned to the Berezin Laboratory at Washington University School of Medicine (St. Louis, Mo.). Berezin Lab was one of a few facilities with an available spectrofluorometer having a spectral range far enough into the near-IR region to produce high-resolution data needed to confirm the original data, upon which the 2-wavelength ratiometric method and system were based.

It is expected that the two calibration curves will be different in terms of noise level and resolution, because the Berezin device effectively has a single optical pathway (channel) and uses the same detector to acquire all wavelength data, whereas the prototype system has two separate pathways and uses two different detectors, as well as two different signal amplifiers, etc. Both devices are capable of producing calibration curves that meet the minimum characteristics as described above in this specification, even if the curves look different.

As comparison of the curve in FIG. 20 and the curve in FIG. 21 (with crosses as data points) demonstrate, they are quite different: although both are monotonic, their slopes are opposite, and the ratio range of the Berezin data is about five times greater. But neither of these invalidates the curve from the experimental system. In fact, by normalizing the ratio scale of the Berezin curve to that of the experimental system (which offsets differences associated with signal intensity and amplification) and by changing the direction of the Berezin data ratio scale (flipping the curve, thereby changing its slope), the resulting curve in FIG. 22 (with circles as data points) closely matches that of the prototype system, as illustrated in FIG. 22 .

The transform function used to normalize the Berezin data ratio scale was as follows: R ₁=(R ₀/5.796)+1.858 where:

R₀ is the original data point value on the Berezin Ratio scale;

R₁ is the transformed point value to be plotted on the prototype device Ratio scale;

5.796 is how much larger the Berezin Ratio data range (difference between the 2- and 16-μM Ratio values) and that for the prototype device; and 1.858 is the amount by which R1 for the 16-μM Berezin sample was shifted on the experimental device's scale (after the Berezin curve was flipped) in order to make it correspond to the location of the experimental device's 2-μM data point.

The computer-generated second-order polynomial curve (labeled “Power”) for the prototype system data does not fit well; this is underscored by the skewness with which it pass through the spread of “+” data points for both the 8- or 16-μM concentrations. Nevertheless, significantly the transformed and flipped “Power” curve for the Berezin data lies entirely within the average-variability envelope of the prototype device's “Power” curve, as delineated by the spreads of data points. This demonstrates that the absolute values of the inter-point relationships for both the Berezin and experimental device data are essentially the same. Therefore, by the best measure currently available, the hard- and soft-ware for determination of ICG concentration in blood appear to be optimized and adequate for human subject evaluation.

Example #4: Approximation Method for Fluorescence Agent Concentration Estimation

In another variation, a method of assessing blood flow and/or tissue perfusion utilizes an approximation of fluorescence agent concentration in the blood flowing through the tissue volume. An intravenous injection of a known amount of ICG dye is administered to a subject, followed by acquisition of a sequence of high-speed angiograms at approximately 20 frames per second for a sequence duration of approximately 15 seconds. The sequence of high-speed angiograms commences at approximately 90 seconds after injection of the ICG dye. It is assumed that after 90 seconds, the injected ICG dye is thoroughly diluted in the circulating blood volume of the subject. In some variations, where the imaging agent has been previously administered to the subject, the approximation method may not involve the administration step.

An approximation of the circulating blood volume of the subject is made after the method disclosed by Nadler et al. (Prediction of blood volume in normal human adults, Surgery 51:224-232 (1962): Man: BV=0.3669×h3+0.03219×w+0.6041 Woman: BV=0.3561×h3+0.03308×w+0.1833

-   -   and where:     -   h: Body height, in meters     -   w: Body weight, in kilograms     -   BV: Body Total Blood Volume, in liters

Concentration of ICG dye is estimated by dividing the amount of ICG dye injected (mg) by the circulating blood volume (ml) estimated by the Nadler formula. This estimated concentration of ICG dye, expressed in is used as the value for C in the modified Beer-Lambert law as described above, to estimate thickness increase in the blood volume layer, ΔL: ΔL=L _(p) −L _(m)=ln[(I _(e) Φ−I _(m))/(I _(e) Φ−I _(p))](εC)⁻¹

After generating a value for ΔL, the blood flow in the tissue volume may be assessed according to F=(A)(ΔL) (P_(DC))/Δt as described above.

Example #5: Comparison of FM-PPG with Radio-Labeled Microspheres

Due to a dearth of published data regarding absolute blood flow levels through various tissues, validation of the tissue perfusion analysis program is difficult beyond rudimentary bench-top experiments. There being no available data regarding readily accessible tissue for comparison, proof-of-concept of the algorithm and experimental device in living tissue heretofore has been in connection with analysis of angiogram data acquired from the medial forearm.

The only blood flow data rendered in absolute terms from tissue accessible without invasive procedures that might serve as a gold standard for comparison was derived from rhesus monkey ocular tissues by Alm and Bill (Exp. Eye Res. 15: 15-29, 1973). These data were derived from, using the well-established method of radiolabeled microsphere injection, are rendered in terms of mg/min/mm². Conversion of their data to μL/sec/mm² requires knowing only that the average density of blood is 1.06×103 kg/m³, the following relationship is easily derived: X(mg/min/mm²)/63.6=X (μL/sec/mm²)

For example, for acquisition of angiographic data from ocular tissue, a fundus camera may be used. However, a fundus camera has no provision for simultaneous acquisition of the additional two wavelengths of data needed for ratiometric determination of the concentration of ICG in circulating blood (C). However, there are alternative ways to determine when during the transit of an ICG bolus, a concentration of 32.2 μM was reached, and a short sequence of images recorded at that time could be analyzed by the FM-PPG algorithm, the results of which can be compared to that of Alm and Bill.

One alternative method is based on concentration fluorescence quenching, a phenomenon exhibited by ICG dye in solution, wherein the fluorescence intensity emitted by the solution increases along with dye concentration until a point is reached beyond which further concentration increase results in fluorescence diminution. For ICG in blood, maximum fluorescence occurs at a concentration of 0.025 mg/ml; above or below this concentration, fluorescence diminution occurs fairly sharply. As an injected ICG bolus transits a network of blood vessels and dye concentration increases, ICG fluorescence also increases and reaches a maximum intensity when the dye concentration reaches 0.025 mg/ml. As concentration continues to increase, fluorescence decreases due to concentration fluorescence quenching, reaching a minimum intensity when concentration reaches its maximum. Thereafter, concentration decreases, causing fluorescence intensity to increase, until it again reaches the maximum level of 0.025 mg/ml; then as concentration continues to decrease, fluorescence also begins to decrease again. Thus, in a plot of overall image brightness versus image number for a sequence of ICG fundus angiograms in which quenching occurred would contain distinctive double peaks of equal fluorescence intensity. Such quenching can be induced in the ocular vasculatures by injecting an ICG bolus of sufficiently high concentration and volume integrity, but only if the dye injection and an immediately following saline flush of proper volume are made rapidly.

A second alternative is based on previously determined amounts of dilution that cubital vein injected dye boluses undergo during transit to the ocular blood vessels (Invest. Ophthal. 12:881-895, 1973): 310 times in an average adult rhesus monkey, and 600 times in an average adult human. Again, a plot of overall image brightness versus image number for a sequence of ICG fundus angiograms can be used, this time simply to determine the subset of angiogram images that were acquired during peak brightness. The peak brightness is then associated with an ICG concentration 1/600 (in human) or 1/310 (in rhesus) that of the injected dye bolus concentration.

In this example, the subject is the dilated right eye of an anesthetized 8.79-kg rhesus monkey (nearly identical to cynomolgus monkeys). Three angiographic sequences were recorded, as follows:

-   -   Sequence 1—saphenous vein injection of 0.1 ml (25 mg/0.7 ml ICG         solution) followed immediately by a rapid 3.0 ml saline flush.     -   Sequence 2—3.0 minutes later with no additional dye injection     -   Sequence 3—3.0 minutes later, saphenous vein injection of 0.1 ml         (25 mg/0.5 ml ICG solution) followed immediately by a rapid 3.0         ml saline flush.

For analysis of each sequence, a Time Plot of image brightness (Total Intensity) versus image number was constructed for the first sequence, as shown in FIG. 23 . From that plot, it was determined the subsequence of images 490-565 were at peak brightness. Since no double peak was detected, the second alternative method for determining the concentration of ICG in circulating blood, as previously described above, was used. That is, the concentration of the first ICG bolus injected was 25 mg/0.7 ml=35.7 mg/ml, which was diluted by 310 times, to a concentration of 0.115 mg/ml during its transit to the eye. Therefore, the peak concentration of dye in the ocular vessels (corresponding to images 490-565) was 148 μM; that value was entered into the appropriate box on the analysis window in lieu of having 2-wavelength ratiometric data available.

A second plot was generated for images 490-565 was generated, and the valleys between two consecutive blood flow pulses were selected (green squares). At the same time, a table indicating computed blood flow for each pulse, as well as average flow (0.058 μL/sec/mm²) was also generated as is shown in FIG. 24 . Generally, the same procedure was followed for the second angiogram sequence shown in FIG. 25 . From this plot, the subset of images 300-381 was selected, and the corresponding ICG dye concentration (C) was calculated based on the intensity of the pulses in this subset (0.0204), compared to the intensity of the peak brightness in the first sequence (0.0595, which corresponded to a 148 μM concentration). Thus, 148 μM/0.0595=C/0.0204, so C=50.7 μM.

The second plot was generated for images 300-381 was generated, and the valleys between five consecutive blood flow pulses were selected (squares). At the same time a table indicating computed blood flow for each pulse, as well as average flow (0.055 μL/sec/mm²) was also generated as is shown in FIG. 26 . Finally, the same procedure was applied to the third sequence, yielding the plots and table, wherein the plot is shown is FIG. 27 .

Again, from that plot, it was determined the subsequence of images 260-290 were at peak brightness, and the concentration of ICG in circulating blood after dilution in transit was calculated. This time, the concentration of the ICG bolus injected was 25 mg/0.5 ml=50.0 mg/ml, which was diluted by 310 times, to a concentration of 0.161 mg/ml during its transit to the eye. Therefore, the peak concentration of dye in the ocular vessels (corresponding to images 260-290) was 207 μM.

The second plot was generated for images 260-290 was generated, and the valleys between two consecutive blood flow pulses were selected (green squares). At the same time the table indicating computed blood flow for each pulse, as well as average flow (0.105 μL/sec/mm²) was also generated as is shown in FIG. 28 .

The above analysis of three consecutive angiograms from the same eye yielded choroidal blood flows of 0.058, 0.055, and 0.105 μL/sec/mm², all of which compare favorably with the gold standard flow of 0.0866 μL/sec/mm², as reported by Alm and Bill and described in the following.

The left-hand image in FIG. 29A is an image from the human eye angiogram. In the right-hand image (FIG. 29B), the retinal area shown in the left-hand image (FIG. 29A) is represented as a box superimposed upon the autoradiograph of a flat-mounted choroid from the left eye of one of the monkeys used by Alm and Bill in their experiments. The hole in the middle is due to the removal of the optic nerve. The black spots represent the trapped microspheres, and the density of the spots is a measure of blood flow rate. The areas encompassed by the red box include the foveal and peripapillary regions, which according to their results from 17 subjects, have blood flows of 6.49 and 4.53 mg/min/mm², respectively. These data translate into an average flow rate of 0.866 μL/sec/mm² for the encompassed area.

Example #6: Comparison of ICG Concentration Estimate with Control Data

As described above, one variation of the FM-PPG method for facilitating assessment of blood flow and/or perfusion includes estimating the instantaneous molar concentration of the fluorescence agent in the tissue volume based on a predetermined amount of the fluorescence agent and an estimated circulating blood volume in the subject. This estimation involves the underlying assumption that after a certain amount of time has elapsed since administration of the fluorescence agent, there is approximately uniform molar concentration of the fluorescence agent throughout the circulating blood volume of the subject (and within the blood flow and perfusion in the tissue volume).

To validate this underlying assumption, an estimated value for molar concentration of a fluorescence agent in a tissue volume for an approximately 150 lb male human subject was compared against control data for the circulating blood volume for the subject. The control data was calibrated against a reference curve as follows. The reference curve was generated by drawing ten blood samples of a subject (without ICG administration), centrifuging the blood samples to remove red blood cells such that the samples had a hematocrit level of approximately 0.54, mixing the plasma of each blood sample with a different, known concentration of ICG to create ICG concentration reference samples, and measuring with an absorption spectrometer the respective absorption of 805 nm light by the plasma in each ICG concentration reference sample. As shown in FIG. 33A, a reference curve was generated based on the absorptivity measurements, which could serve as a look-up table to correlate absorptivity of the 805 nm light to ICG concentration. In some variations, e.g., as an alternative to measurement with an absorption spectrometer, the plasma and ICG sample absorption at 805 nm may be measured using a narrow band light source of wavelength about 805 nm and a photodetector, such as a photodiode, placed on the side of the sample opposite to the light source.

Approximately 0.3 cc of ICG dye having a concentration of approximately 25 mg/ml (approximately 9.7 moles of ICG) was administered to one arm of a subject. To generate the control data, following the injection of ICG, a series of blood samples was drawn from the opposing arm of the subject over time (approximately t=20 s, 50 s, 75 s, 145 s, 310 s, and 575 s after ICG administration) to give sampling of ICG concentration in the circulating blood volume of the subject over time. These samples were then centrifuged to separate the red blood cells from the plasma and the plasma layer (containing the ICG) was decanted off. The absorption of 805 nm light by each plasma sample was measured with an absorption spectrometer, and these measurements were compared to the reference curve of FIG. 33A to determine ICG concentration of each sample (that is, for each sampled point in time). In some embodiments, ex-vivo absorption measurements could, alternately or additionally, be performed using a narrow band source having a wavelength at or near 805 nm, and a photodetector such as a photodiode. FIG. 33B shows, as control data, the determined ICG concentration of the six blood samples over time following ICG administration, which includes a correction for the subject's hematocrit level of 0.46 instead of 0.54 associated with the reference curve. As shown in FIG. 33B, there is a plateau or steady-state molar concentration of ICG of around 1 μM at around 200 seconds after ICG administration.

Based on the same ICG administration event, the instantaneous molar concentration of ICG in the tissue volume of the subject was estimated based on the predetermined amount of administered ICG and an estimated circulating blood volume of the patient. Approximately 9.7 moles of ICG were injected, while an approximately 150 lb male human subject has an estimated circulating blood volume of about 77 milliliters of blood per kilogram of body mass. Based on these numbers, the instantaneous molar concentration of ICG in the tissue volume was estimated to be approximately 1.48 which is similar to the steady-state molar concentration of ICG shown in the control data of FIG. 33B for circulating blood volume of the subject.

Example #7: Comparison of Estimated ICG Concentration and ICG Concentration Determined by Ratiometric Method

Approximately 1 cc of ICG, diluted in water and having a concentration of 2.5 mg/ml, was administered to a pig, such that the pig received approximately 3.23 μM of ICG. The pig, having a mass of approximately 68 kg, had a blood volume of approximately 3.13-503 L of blood. Signal intensity of fluorescence emitted from the ICG in the tissue volume at a short wavelength (SWL) and a long wavelength (LWL) after excitation is shown in FIG. 34A.

Applying the ratiometric method to the signal intensities in the SWL and LWL spectral bands results in a determined ICG concentration at full dilution of between approximately 0.6-1 μM, as indicated by the plateau in FIG. 34B. Applying an estimation method of ICG concentration to the known values of 3.23 μM of ICG and 3.13-5.03 L of circulating blood volume in the pig results in an estimated ICG concentration of approximately 3 μM. These two methods yield results in ICG concentration at full dilution that are within the same order of magnitude, and can be used alone or in combination to facilitate assessment of blood flow and/or tissue perfusion in the tissue volume.

Example #8: Comparison of Estimated ICG Concentration and ICG Concentration Determined by Ratiometric Method

Approximately 3.2 μM of ICG was administered to a 28 kg pig having a total circulating blood volume of about 1.29-2.07 L. The ICG was excited and measurements of mean fluorescence signal intensity at a short wavelength (SWL) in Channel 1 and a long wavelength (LWL) in Chanel 2 were plotted over time, as shown in FIG. 35 . The delay between t=0 and approximately t=10 sec is due to the long IV line used to administer the ICG injection.

Applying the ratiometric method to the signal intensities in the SWL and LWL spectral bands results in a determined ICG concentration at full dilution. As shown in FIG. 35 , the plot of concentration of blood-borne ICG as a function of time shows typical noisy characteristics prior to the arrival of the ICG bolus into the area of interest. The noisy data prior to the onset of fluorescence is due to the low signal to noise ratio in both the channels. Once there is sufficient fluorescence to make a meaningful measurement, the concentration increases rapidly to around 7 then decays rapidly during a dilution phase. At about t=35 sec, the ICG concentration at full dilution is about 1 μM. Subsequently, the concentration further decays at a less-rapid rate as the dye is diluted and metabolized within the body by the liver. As the total fluorescence decays, the ratiometric measurement of concentration becomes noisier again, but becomes centered around zero concentration.

Applying an estimation method of ICG concentration to the known values of 3.2 μM of ICG and 1.29-2.07 L of circulating blood volume in the pig results in an estimated ICG concentration of approximately 1.5-2.5 μM. These two methods, yielding similar estimates of ICG concentration at full dilution of 1 μM and 1.5-2.5 respectively, can be used alone or in combination to facilitate assessment of blood flow and/or tissue perfusion in the tissue volume.

While the present disclosure has been illustrated and described in connection with various embodiments shown and described in detail, it is not intended to be limited to the details shown, since various modifications and structural changes may be made without departing in any way from the scope of the present disclosure. Various modifications of form, arrangement of components, steps, details and order of operations of the embodiments illustrated, as well as other embodiments of the disclosure may be made without departing in any way from the scope of the present disclosure, and will be apparent to a person of skill in the art upon reference to this description. It is therefore contemplated that the appended claims will cover such modifications and embodiments as they fall within the true scope of the disclosure. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the disclosure includes embodiments having combinations of all or some of the features described. For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. 

What is claimed is:
 1. A method for use in medical imaging for facilitating assessment of microvascular tissue perfusion in a tissue volume of a subject, the method comprising: after a predetermined amount of a fluorescence agent has been administered to the subject, exciting the fluorescence agent in the tissue volume such that the excited fluorescence agent emits fluorescent light; acquiring fluorescence image data based on the fluorescent light emitted during blood flow through the tissue volume; processing the fluorescence image data to compensate for ambient light; processing the fluorescence image data by suppressing image data corresponding to non-microvascular blood vessels; and generating an assessment of microvascular tissue perfusion in the tissue volume based at least in part on the processed fluorescence image data.
 2. The method of claim 1, wherein: the method further comprises determining a molar concentration of the fluorescence agent in the blood flowing through the tissue volume; and generating the assessment of microvascular tissue perfusion in the tissue volume is based at least in part on the molar concentration.
 3. The method of claim 2, wherein determining the molar concentration comprises estimating the molar concentration.
 4. The method of claim 3, wherein estimating the molar concentration comprises estimating a circulating blood volume in the subject.
 5. The method of claim 4, wherein estimating the circulating blood volume in the subject is based at least in part on one or more of sex, body height, and body weight of the subject.
 6. The method of claim 2, wherein determining the molar concentration comprises calculating the molar concentration.
 7. The method of claim 6, wherein calculating the molar concentration is based at least in part on a spectral shift in a fluorescence emission spectrum of the fluorescence agent.
 8. The method of claim 1, wherein generating the assessment of microvascular tissue perfusion in the tissue volume is based at least in part on one or more of a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.
 9. The method of claim 1, wherein generating an assessment of microvascular tissue perfusion is based at least in part on an intensity of the fluorescence image data during a diastolic phase of blood flow and an intensity of the fluorescence image data during a systolic phase of the blood flow.
 10. The method of claim 1, wherein the tissue volume is selected in the fluorescence image data by a user.
 11. The method of claim 1, wherein the fluorescence agent comprises indocyanine green.
 12. A system for facilitating assessment of microvascular tissue perfusion in a tissue volume of a subject, the system comprising: a light source configured to excite a fluorescence agent in the tissue volume, after a predetermined amount of the fluorescence agent has been administered to the subject, such that the fluorescence agent emits fluorescent light; one or more processors; and memory having instructions stored thereon, wherein the instructions, when executed by the one or more processors, cause the system to: receive fluorescence data based on the fluorescent light emitted from the excited fluorescence agent in the tissue volume; process the fluorescence image data to compensate for ambient light; process the fluorescence image data by suppressing image data corresponding to non-microvascular blood vessels; and generate an assessment of microvascular tissue perfusion in the tissue volume based at least in part on the processed fluorescence image data and the determined concentration of the fluorescence agent.
 13. A non-transitory computer-readable storage medium storing instructions for facilitating assessment of microvascular tissue perfusion in a tissue volume of a subject, wherein the instructions, when executed by a system comprising a light source and one or more processors, cause a system to: excite, by the light source, a fluorescence agent in the tissue volume, after a predetermined amount of the fluorescence agent has been administered to the subject, such that the fluorescence agent emits fluorescent light; receive fluorescence image data based on the fluorescent light emitted during blood flow through the tissue volume; process the fluorescence image data to compensate for ambient light; process the fluorescence image data by suppressing image data corresponding to non-microvascular blood vessels; and generate an assessment of microvascular tissue perfusion in the tissue volume based at least in part on the processed fluorescence image data and the determined concentration of the fluorescence agent.
 14. The system of claim 12, wherein: the method further comprises determining a molar concentration of the fluorescence agent in the blood flowing through the tissue volume; and generating the assessment of microvascular tissue perfusion in the tissue volume is based at least in part on the molar concentration.
 15. The system of claim 14, wherein determining the molar concentration comprises estimating the molar concentration.
 16. The system of claim 15, wherein estimating the molar concentration comprises estimating a circulating blood volume in the subject.
 17. The system of claim 16, wherein estimating the circulating blood volume in the subject is based at least in part on one or more of sex, body height, and body weight of the subject.
 18. The system of claim 14, wherein determining the molar concentration comprises calculating the molar concentration.
 19. The system of claim 18, wherein calculating the molar concentration is based at least in part on a spectral shift in a fluorescence emission spectrum of the fluorescence agent.
 20. The system of claim 12, wherein generating the assessment of microvascular tissue perfusion in the tissue volume is based at least in part on one or more of a cross-sectional area of the tissue volume, a thickness increase of a blood volume layer, a pulse duty cycle of blood flow, and duration of a single pressure pulse in blood flow.
 21. The system of claim 12, wherein generating an assessment of microvascular tissue perfusion is based at least in part on an intensity of the fluorescence image data during a diastolic phase of blood flow and an intensity of the fluorescence image data during a systolic phase of the blood flow.
 22. The system of claim 12, wherein the tissue volume is selected in the fluorescence image data by a user.
 23. The system of claim 12, wherein the fluorescence agent comprises indocyanine green. 